CN117916263A - Anti-TIGIT antibodies and uses thereof - Google Patents

Abstract

The present invention relates to: novel anti-TIGIT antibodies; and their use for enhancing immunity, anticancer therapy and prevention and/or treatment of immune diseases.

Description

Anti-TIGIT antibodies and uses thereof

Technical Field

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of KR 10-2021-0060014, filed on May 10, 2021, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

Disclosed are novel anti-TIGIT antibodies and their use for immune enhancement and for preventing and/or treating cancer and immune-related diseases.

Background Art

Cancer immunotherapy is a method of treating cancer by using the body's immune response, and is known as the fourth cancer treatment method after chemotherapy, surgery, and radiotherapy. Cancer immunotherapy requires the activation of the immune system's mechanisms to increase recognition and response to tumor cells. The activation of the immune system involves complex mechanisms, including the functions of various cells, such as antigen-presenting cells, which are essential for initiating antigen-specific responses, and effector cells, which are responsible for destroying tumor cells.

Representative of effector cells are cytotoxic T cells.

Meanwhile, T cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibitory motif) domain (TIGIT), also known as WUCAM, VSIG9 or Vstm3, is a co-inhibitory receptor that is preferentially expressed not only on NK, CD8+ and CD4+ T cells, but also on regulatory T cells (Treg). TIGIT is a transmembrane protein that contains an intracellular ITIM domain, a transmembrane domain and an immunoglobulin variable domain.

TIGIT expression is elevated in tumor infiltrating lymphocytes (TILs) and in disease states such as infection. TIGIT expression can serve as a marker for exhausted T cells with reduced effector function compared to TIGIT-negative cells. In addition, Treg cells expressing TIGIT display enhanced immunosuppressive activity compared to TIGIT-negative Treg populations.

Based on these findings, drugs that counteract TIGIT (e.g., antagonistic anti-TIGIT antibodies) are expected to induce immune system activation and enhance immune responses in disease states such as cancer, thereby exhibiting beneficial therapeutic effects.

Summary of the invention

Technical issues

Provided are anti-TIGIT antibodies that bind to TIGIT and their pharmaceutical uses.

In one aspect, an anti-TIGIT antibody or antigen-binding fragment thereof that binds to TIGIT is provided.

The anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

a heavy chain complementarity determining region consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (CDR-H3), or a heavy chain variable region comprising the heavy chain complementarity determining region; and

A light chain complementary determining region consisting of a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 5, and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 6, or a light chain variable region comprising the light chain complementary determining region.

In embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; and

A light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

On the other hand, provided are immunopotentiators or immunopotentiating pharmaceutical compositions, each comprising an anti-TIGIT antibody or an antigen-binding fragment thereof as an active ingredient.

On the other hand, provided is a pharmaceutical composition for preventing and/or treating immune-related diseases, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof as an active ingredient.

On the other hand, provided are anticancer agents or pharmaceutical compositions for preventing and/or treating cancer, each comprising an anti-TIGIT antibody or an antigen-binding fragment thereof as an active ingredient.

On the other hand, a method for immune enhancement is provided, which comprises the step of administering a pharmaceutically effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof to a subject in need of immune enhancement.

On the other hand, a method for preventing and/or treating an immune-related disease is provided, which comprises the step of administering a pharmaceutically effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof to a subject in need of prevention and/or treatment of an immune-related disease.

Another aspect provides a method for preventing and/or treating cancer, comprising the step of administering a pharmaceutically effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof to a subject in need of cancer prevention and/or treatment.

On the other hand, provided is the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for immune enhancement or for preparing an immune enhancer.

On the other hand, provided is the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for preventing and/or treating immune-related diseases or for preparing a medicament for preventing and/or treating immune-related diseases.

On the other hand, provided is the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for preventing and/or treating cancer or for preparing a medicament for preventing and/or treating cancer.

According to the embodiment, in the pharmaceutical compositions, methods and uses provided herein, anti-TIGIT antibodies or antigen-binding fragments thereof can be used in combination with immune checkpoint proteins, such as drugs (antagonists) targeting one or both of PD-1 and PD-L1. The drugs that can be co-administered can be anti-PD-1 antibodies, anti-PD-L1 antibodies, or both, but are not limited thereto.

In another aspect, a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region or a heavy chain of an anti-TIGIT antibody is provided.

In another aspect, a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or a light chain of an anti-TIGIT antibody is provided.

On the other hand, a recombinant vector is provided, which carries a combination of a nucleic acid molecule encoding a heavy chain complementary determining region, a heavy chain variable region or a heavy chain of an anti-TIGIT antibody and a nucleic acid molecule encoding a light chain complementary determining region, a light chain variable region or a light chain of an anti-TIGIT antibody, or a separated recombinant vector, which carries a nucleic acid molecule encoding a heavy chain complementary determining region, a heavy chain variable region or a heavy chain of an anti-TIGIT antibody and a nucleic acid molecule encoding a light chain complementary determining region, a light chain variable region or a light chain of an anti-TIGIT antibody, respectively. The recombinant vector can be an expression vector for expressing a nucleic acid molecule.

Another aspect provides a recombinant cell harboring a recombinant vector.

On the other hand, a method for producing an anti-TIGIT antibody or an antigen-binding fragment thereof is provided, the method comprising the step of expressing a nucleic acid molecule in a host cell. The step of expressing a nucleic acid molecule may be a step of culturing a cell anchoring the nucleic acid molecule or a recombinant vector carrying the nucleic acid molecule. The method may also include a step of isolating and/or purifying the antibody from the culture medium after the expression step.

Technical Solution

Provided herein are anti-TIGIT antibodies or antigen-binding fragments thereof that bind to TIGIT and their pharmaceutical uses. Anti-TIGIT antibodies or antigen-binding fragments thereof activate immunity by blocking the action of TIGIT (e.g., enhancing effector T cell function, regulating Treg activity, increasing cytokine secretion, etc.), thereby being applied to various immune activators and/or immunotherapies.

Hereinafter, a detailed description of the present disclosure will be given.

Antibodies or antigen-binding fragments

In one aspect, an anti-TIGIT antibody or antigen-binding fragment thereof that binds to TIGIT is provided.

The anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1),

a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 2,

a polypeptide (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 3,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1),

A polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and

A polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3).

The polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO:4 may comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

Complementarity determining regions (CDRs) are generally defined herein according to the Kabat system.

In an embodiment, the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that may be included in the anti-TIGIT antibodies or antigen-binding fragments thereof provided herein are summarized in Table 1 below.

Table 1

(In Table 1, CDR-H1, CDR-H2 and CDR-H3 each represent a heavy chain complementarity determining region, and CDR-L1, CDR-L2 and CDR-L3 each represent a light chain complementarity determining region)

In embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, and CDR-H3 of SEQ ID NO: 3, and

A light chain variable region comprising CDR-L1 of SEQ ID NO:4 (i.e., SEQ ID NO:7 or SEQ ID NO:8), CDR-L2 of SEQ ID NO:5, and CDR-L3 of SEQ ID NO:6.

More specifically, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14; and

A light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

In embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18;

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19; or

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO:14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20.

Depending on the circumstances (eg, when recombinantly produced), the heavy chain variable region and/or the light chain variable region may also contain an appropriate signal sequence at the N-terminus.

The amino acid sequences of the heavy chain variable region and the light chain variable region that may be contained in the anti-TIGIT antibodies or antigen-binding fragments thereof provided herein are given in Table 2 below:

Table 2

(In Table 2, the underlined regions represent CDR1, CDR2, and CDR3 in the heavy chain and light chain, respectively)

In an embodiment, the anti-TIGIT antibodies or antigen-binding fragments thereof provided herein bind to a TIGIT protein, for example, a region selected from amino acid residues 51 to 70 of the human TIGIT protein (NCBI reference sequence NP_776160.2; UniProtKB/SwissProt Q495A1-1) (TAQVTQVNWEQQDQLLAICN; SEQ ID NO: 31), but are not limited thereto.

[Human TIGIT protein (SEQ ID NO: 30)]

As described herein, the term "antibody" may refer to a protein that specifically binds to a specific antigen, and may be a protein produced by stimulating an antigen in the immune system, or a protein produced by chemical synthesis or recombinant production, without specific limitations. The antibody may be non-naturally occurring, for example, produced by recombination or synthesis. The antibody may be an animal antibody (e.g., a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal or polyclonal antibody.

In the anti-TIGIT antibodies or antigen-binding fragments thereof provided herein, the portions other than the heavy chain CDR and light chain CDR portions or the heavy chain variable regions and light chain variable regions defined above can be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.), and, for example, derived from framework portions, and/or light chain constant regions and/or heavy chain constant regions. In embodiments, the anti-TIGIT antibodies provided herein can be antibodies in the form of human IgG, such as IgG1, IgG2, IgG3, or IgG4, but are not limited thereto.

A complete antibody (e.g., IgG type) has a structure of two full-length light chains and two full-length heavy chains, wherein each light chain is connected to the corresponding heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region is of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), or alpha2 (α2) as its subclass. The light chain constant region is of kappa (κ) or lambda (λ) type.

In an embodiment, the anti-TIGIT antibody provided herein may comprise an IgG constant region as a heavy chain constant region and a κ constant region as a light chain constant region, but is not limited thereto.

In an embodiment, the constant region of IgG (e.g., human IgG1) can be a wild type. In another embodiment, the constant region of IgG can be a variant having at least one mutation on human IgG1, wherein the mutation is selected from S240D (S at position 240 is replaced by D, and amino acid mutations are expressed in the same manner hereinafter), A331L, I333E, N298A, S299A, E334A, K335A, L235A, L236A, and P330G. For example, the constant region can be a variant having the following mutations:

(1) S240D, A331L and I333E;

(2) N298A;

(3) S299A, E334A, and K335A; or

(4) L235A, L236A and P330G.

As used herein, the term "heavy chain" may include full-length heavy chains and fragments thereof, wherein a full-length heavy chain comprises a variable region _VH comprising an amino acid sequence sufficient to provide antigen specificity, three constant regions _CH1 , _CH2 and _CH3 , and a hinge. The term "light chain" is intended to include full-length light chains and fragments thereof, wherein a full-length light chain comprises a variable region _VL comprising an amino acid sequence sufficient to provide antigen specificity and a constant region _CL .

The term "complementarity determining region" (CDR) refers to the portion of the antibody variable region that confers antigen binding specificity, and refers to the amino acid sequence found in the hypervariable region of the immunoglobulin heavy chain or light chain. The heavy chain and the light chain can each contain three CDRs (CDRH1, CDRH2 and CDRH3 and CDRL1, CDRL2 and CDRL3). CDRs can provide contact residues that play an important role in the binding of an antibody to its antigen or antigenic epitope. As used herein, the terms "specific binding" and "specific recognition" may have the same general meaning as known to those of ordinary skill in the art, and indicate that antibodies and antigens specifically interact with each other to cause an immune response.

The complementarity determining regions (CDRs) described herein are defined according to the Kabat system.

In the present disclosure, unless otherwise specified, the term "antibody" may be understood to include an antigen-binding fragment of an antibody having antigen-binding ability.

The term "antigen-binding fragment" used herein may refer to any type of polypeptide comprising a portion capable of binding to an antigen (e.g., 6 CDRs defined herein), and may be, for example, scFv, scFv-Fc, (scFv) ₂ , Fab, Fab' or F(ab') ₂ , but is not limited thereto.

Among the antigen-binding fragments, Fab has a structure consisting of light chain and heavy chain variable regions, a light chain constant region and the first constant region of the heavy chain ( _CH1 ), retaining one antigen-binding site.

Fab' differs from Fab in that Fab' comprises a hinge region with at least one cysteine residue at the C-terminus of the heavy chain _CH1 domain.

F(ab') ₂ antibodies are formed by disulfide bridges of cysteine residues in the Fab' hinge region. Fv is the smallest antibody fragment consisting only of the heavy chain variable region and the light chain variable region. Recombinant techniques for producing Fv fragments are well known in the art.

Double-chain Fv comprises a heavy chain variable region and a light chain variable region mutually linked by non-covalent bonds. Single-chain Fv usually comprises a heavy chain variable region and a light chain variable region, which are mutually linked by covalent bonds through a peptide linker, or are directly linked at the C-terminus to have a dimer structure similar to a double-chain Fv.

The antigen-binding fragment can be obtained using a protease (for example, Fab can be obtained by restrictive cleavage of a whole antibody with papain, and a F(ab') ₂ fragment can be obtained by cleavage with pepsin), or can be prepared by using genetic recombination technology.

The term "hinge region" refers to the region between the _CH1 and _CH2 domains in an antibody heavy chain, which functions to provide flexibility to the antigen binding site in the antibody.

The antibodies provided herein can be monoclonal antibodies. Monoclonal antibodies can be prepared using methods known in the art, such as using phage display technology. Alternatively, antibodies can be constructed in the form of monoclonal antibodies derived from animals (e.g., mice) by conventional methods.

At the same time, based on the binding potential against the TIGIT receptor binding domain, individual monoclonal antibodies can be screened using a typical ELISA (enzyme-linked immunosorbent assay) format. The inhibitory activity can be verified by functional assays, such as competitive ELISA for verifying the molecular interactions of the binding components, or functional assays, such as cell-based assays. Then, for the monoclonal antibody members selected based on their strong inhibitory activity, their affinity (Kd value) to the receptor binding domain of TIGIT can be verified separately.

The final selected antibodies can be prepared and used as humanized antibodies and human immunoglobulin antibodies, in which the remaining parts except the antigen binding portion are humanized. Methods for producing humanized antibodies are well known in the art.

The antigen-binding fragment of the anti-TIGIT antibody provided herein may refer to a fragment derived from an anti-TIGIT antibody and retaining the antigen (TIGIT) binding affinity of the anti-TIGIT antibody. In an embodiment, the antigen-binding fragment may be a polypeptide comprising the 6 CDRs of an anti-TIGIT antibody as described above, and may be, for example, scFv, scFv-Fc, scFv-Ck (κ constant region), scFv-Cλ (λ constant region), (scFv) ₂ , Fab, Fab′ or F(ab′) ₂ , but is not limited thereto. In an embodiment, the antigen-binding fragment may be a fusion polypeptide in which the scFv or scFv is fused to the Fc region (scFv-Fc) or the light chain constant region (eg, κ or λ) (scFv-Ck or scFv-Cλ) of an immunoglobulin (eg, IgG1, IgG2, IgG3, IgG4, etc.), but is not limited thereto.

In the present disclosure, an antibody (e.g., CDR, variable region, or heavy/light chain, antigen-binding fragment, etc.) "comprises (contains) a specific amino acid sequence or consists of a specific amino acid sequence" refers to all cases in which the amino acid sequence is substantially included, and/or insignificant mutations (e.g., substitution, deletion and/or addition of amino acid residues) that do not affect the activity of the antibody (e.g., antigen-binding affinity, pharmacological activity) are introduced into the amino acid sequence.

The anti-TIGIT antibodies or antigen-binding fragments thereof provided herein may have a binding affinity ( _KD ) for TIGIT (e.g., human TIGIT) of 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 0.1 mM or less, 0.05 mM or less, 0.01 mM or less, 0.005 mM or less, or 0.001 mM or less. Less than 0.001 mM, for example, 0.0001 nM to 10 mM, 0.0005 nM to 10 mM, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM, 0.0001 nM to 5 mM, 0. 0.0005nM to 5mM, 0.001nM to 5mM, 0.005nM to 5mM, 0.01nM to 5mM, 0.05nM to 5mM, 0.1nM to 5mM, 0.5nM to 5mM, 1nM to 5mM, 0.0001nM to 1mM, 0.0005nM to 1mM, 0.001nM to 1mM, 0.005nM to 1mM, 0.001nM to 1mM, 0.005nM to 1mM, 0 0.01nM to 1mM, 0.05nM to 1mM, 0.1nM to 1mM, 0.5nM to 1mM, 1nM to 1mM, 0.0001nM to 0.5mM, 0.0005nM to 0.5mM, 0.001nM to 0.5mM, 0.005nM to 0.5mM, 0.01nM to 0.5mM, 0.05nM to 0.5mM, 0.1nM to 0.5mM, 0.1nM to 0.5mM M to 0.5 mM, 0.5 nM to 0.5 mM, 1 nM to 0.5 mM, 0.0001 nM to 0.2 mM, 0.0005 nM to 0.2 mM, 0.001 nM to 0.2 mM, 0.005 nM to 0.2 mM, 0.01 nM to 0.2 mM, 0.05 nM to 0.2 mM, 0.1 nM to 0.2 mM, 0.5 nM to 0.2 mM M, 1nM to 0.2mM, 0.0001nM to 0.1mM, 0.0005nM to 0.1mM, 0.001nM to 0.1mM, 0.005nM to 0.1mM, 0.01nM to 0.1mM, 0.05nM to 0.1mM, 0.1nM to 0.1mM, 0.5nM to 0.1mM, 1nM to 0.1mM, 0.0001nM to 0.1mM, 0.005nM to 0.1mM, 0.01nM to 0.1mM, 0.05nM to 0.1mM, 0.1nM to 0.1mM, 0.5nM to 0.1mM, 1nM to 0.1mM, 0.0001nM M to 0.05mM, 0.0005nM to 0.05mM, 0.001nM to 0.05mM, 0.005nM to 0.05mM, 0.01nM to 0.05mM, 0.05nM to 0.05mM, 0.1nM to 0.05mM, 0.5nM to 0.05mM, 1nM to 0.05mM, 0.0001nM to 0.01mM, 0.0005nM to 0.01mM, 0.001nM to 0.01mM, 0.005nM to 0.01mM, 0.01nM to 0.01mM, 0.05nM to 0.01mM, 0.1nM to 0.01mM, 0.5nM to 0.01mM, or 1nM to 0.01mM as measured by surface plasmon resonance (SPR), but not limited thereto.

On the other hand, a polypeptide molecule is provided, which includes a heavy chain complementary determining region (CDR-H1, CDR-H2, CDR-H3 or a combination thereof), a light chain complementary determining region (CDR-L1, CDR-L2, CDR-L3 or a combination thereof), a combination thereof; or a heavy chain variable region, a light chain variable region or a combination thereof in the aforementioned anti-TIGIT antibody.

The polypeptide molecule can be used as an antibody precursor for preparing antibodies, or can be contained in a protein scaffold having an antibody-like structure (eg, peptide antibody, nanobody), bispecific antibody or multispecific antibody as a component thereof (eg, CDR or variable region).

As used herein, the term "peptibody" (peptide + antibody) refers to a fusion protein with a framework and function similar to that of an antibody, in which a peptide is fused to all or part of the constant region of an antibody, such as the Fc region, and serves as an antigen binding site (heavy chain and/or light chain CDR or variable region).

The term "nanoantibody" as used herein is referred to as a single domain antibody, and refers to an antibody fragment comprising a single variable domain of an antibody in monomeric form, which exhibits characteristics of selective binding to a specific antigen similar to an antibody with a complete structure. The molecular weight of a nanoantibody is typically about 12 kDa to about 15 kDa, which is very small compared to the normal molecular weight (about 150 kDa or about 160 kDa) of a complete antibody (including two heavy chains and two light chains), and is smaller than a Fab fragment or a scFv fragment in some cases.

As used herein, the term "multispecific antibody" (including bispecific antibodies) refers to an antibody that recognizes and/or binds to two or more different antigens, or recognizes and/or binds to different sites of the same antigen. One antigen binding site of the multispecific antibody may include the above-mentioned polypeptide, antibody or antigen-binding fragment to bind to TIGIT.

The polypeptide, antibody, or antigen-binding fragment that binds to TIGIT provided herein can be used in the form of a conjugate with at least one selected from a useful polymer, a label, and the like.

Useful polymers can be, for example, non-protein polymers that increase the in vivo half-life of polypeptides, antibodies and/or antigen-binding fragments, and can be at least one hydrophilic polymer selected from polyethylene glycol (PEG) (e.g., 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa PEG), dextran, monomethoxy polyethylene glycol (mPEG), etc., but are not limited thereto.

The label can be at least one radionucleotide or a fluorescent or chemiluminescent small chemical selected from rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanates, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, 152Eu, dansyl, umbelliferone, luciferin, luminol label, isoluminol label, aromatic acridinium ester label, imidazole label, acridinium salt label, oxalate label, aequorin label, 2,3-dihydrophthalazinone, biotin/avidin, spin label and stable free radicals, but is not limited thereto.

Medical Uses

The anti-TIGIT antibodies or antigen-binding fragments thereof provided herein activate immunity (e.g., enhancing effector T cell function, regulating Treg activity, increasing cytokine secretion, etc.) by blocking the action of TIGIT (e.g., the interaction between TIGIT and its ligand CD155 (PVR)). Therefore, anti-TIGIT antibodies or antigen-binding fragments thereof can be used for immune enhancement and can find favorable applications in the prevention and/or treatment of immune-related diseases, especially cancer.

On the other hand, provided are immunopotentiators or immunopotentiating pharmaceutical compositions, each comprising an anti-TIGIT antibody or an antigen-binding fragment thereof as an active ingredient.

On the other hand, provided is a pharmaceutical composition for preventing and treating immune-related diseases, which comprises an anti-TIGIT antibody or an antigen-binding fragment thereof as an active ingredient.

On the other hand, provided are anticancer agents or pharmaceutical compositions for preventing and/or treating cancer, each comprising an anti-TIGIT antibody or an antigen-binding fragment thereof as an active ingredient.

Another aspect provides an immunoenhancement method, which includes administering a pharmaceutically effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof to a subject in need of immunoenhancement. The immunoenhancement method may also include determining a subject in need of immunoenhancement before the administering step.

On the other hand, a method for preventing and/or treating an immune-related disease is provided, the method comprising administering a pharmaceutically effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof to a subject in need of immune-related disease prevention and/or treatment. The method for preventing and/or treating an immune-related disease may also include the step of determining a subject in need of immune-related disease prevention and/or treatment prior to the administering step.

On the other hand, a method for preventing and/or treating cancer is provided, which includes the step of administering a pharmaceutically effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof to a subject in need of cancer prevention and/or treatment. The method for preventing and/or treating cancer may also include the step of determining a subject in need of cancer prevention and/or treatment prior to the administering step.

On the other hand, provided is the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for immune enhancement or for preparing an immune enhancer.

On the other hand, provided is the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for preventing and/or treating immune-related diseases or for preparing a medicament for preventing and/or treating immune-related diseases.

On the other hand, provided is the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for preventing and/or treating cancer or for preparing a medicament for preventing and/or treating cancer.

In the pharmaceutical compositions, methods and uses provided herein, anti-TIGIT antibodies or antigen binding fragments thereof can be used in combination with immune checkpoint proteins, such as drugs (antagonists) targeting one or both of PD-1 and PD-L1. Specifically, in addition to anti-TIGIT antibodies or antigen binding fragments thereof, the pharmaceutical composition may also include drugs targeting one or both of PD-1 and PD-L1. In addition to the step of administering an anti-TIGIT antibody or antigen binding fragment, the method may also include the step of administering a drug targeting one or both of PD-1 and PD-L1.

The drug that can be administered in combination can be an anti-PD-1 antibody, an anti-PD-L1 antibody, or both, but is not limited thereto. In an embodiment, the anti-PD-1 antibody can be at least one selected from pembrolizumab and nivolumab, but is not limited thereto.

As used herein, the term "immune enhancement" (or enhancing immunity) refers to inducing an initial immune response to an antigen or enhancing an existing immune response, and can be used interchangeably with the terms immune stimulation, immune increase, immune activation, etc. In an embodiment, immune enhancement can be achieved by selecting from immune cells (effector T cells, such as cytotoxic T cells; CD3+T cells, CD4+T cells, CD8+T cells, etc.), inactivation and/or depletion of regulatory T (Treg) cells, and increased production and/or secretion of immune proteins (such as cytokines, etc.), but is not limited thereto.

As used herein, the term "immune-related disease" includes all diseases caused by damage and/or insufficient activity of the immune system. Examples of immune-related diseases include cancer, infectious diseases, autoimmune diseases, inflammatory diseases, etc., but are not limited thereto.

The cancer may be a solid cancer or a blood cancer, but is not limited thereto, and may be at least one selected from squamous cell carcinoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), peritoneal cancer, skin cancer, melanoma (e.g., skin or intraocular melanoma, etc.), rectal cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, liver cancer, bile duct cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer (e.g., colon cancer, rectal cancer, colorectal cancer, etc.), endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, head and neck cancer, brain cancer, and osteosarcoma.

The preventive and/or therapeutic effects on cancer include all effects of removing (killing) cancer cells, inhibiting the generation and/or growth of cancer cells, and inhibiting the progression of cancer due to migration, invasion, metastasis, and the like.

Infectious diseases, autoimmune diseases and inflammatory diseases can be selected from the aforementioned immune enhancement (e.g., immune cells (effector T cells, such as cytotoxic T cells; CD3+T cells, CD4+T cells, CD8+T cells, etc.), inhibition and/or depletion of the activity of regulatory T (Treg) cells, immune proteins (e.g., increased production and/or secretion of cytokines (IL-2, IFN-γ, etc.)). For example, infectious diseases are a general term for diseases that occur when pathogens, such as viruses, bacteria, fungi and parasites, spread and invade living bodies (e.g., animals including humans). They can be infections or diseases caused by one or more pathogens selected from viruses, bacteria, fungi, parasites, etc. Autoimmune diseases can be selected from rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, Lupus, Sjögren's syndrome, myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, cytokine storm, etc., but are not limited thereto. The term "inflammatory disease" refers to inflammation (e.g., chronic inflammation or acute inflammation) or a disease caused by inflammation. Examples of inflammatory diseases include cardiac inflammation (e.g., coronary artery disease, angina pectoris, myocardial infarction, pericarditis, myocarditis, etc.), vascular inflammation (e.g., atherosclerosis, vasculitis, disseminated intravascular coagulation (DIC), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), anemia, etc.), upper respiratory tract inflammation (e.g., acute nasopharyngitis, allergic rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, etc.), lower Inflammation of the respiratory tract and/or lungs (e.g., bronchitis, bronchiectasis, asthma, chronic active pulmonary disease (COPD), pneumonia, interstitial lung disease, tuberculosis, etc.), inflammation of the upper gastrointestinal tract (e.g., gastritis, esophagitis, etc.), inflammation of the lower gastrointestinal tract (e.g., enteritis, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel syndrome, appendicitis, perianal fistula, etc.), inflammation of the liver, bile duct and/or pancreas (e.g., hepatitis, fatty liver, cholangitis, cholecystitis, pancreatitis, type 1 diabetes, etc.), inflammation of the kidneys (upper urinary tract) (e.g., pyelonephritis, glomerulonephritis, urinary tract infection, etc.), inflammation of the lower urinary tract (e.g., urinary tract infection, ureteritis, urethritis, cystitis, prostatitis/chronic pelvic pain syndrome, etc.), inflammation of the thyroid and/or parathyroid glands (e.g., thyroiditis, parathyroiditis, etc.), inflammation of the adrenal glands (e.g., adrenal inflammation, etc.), inflammation of the genitals (e.g., , pelvic inflammatory disease, oophoritis, orchitis, epididymitis, etc.), bone and/or joint inflammation (e.g., osteoarthritis, rheumatoid arthritis, osteomyelitis, synovitis, etc.), skin inflammation (e.g., skin cellulitis, erysipelas, tinea versicolor, tinea pedis, acne, etc.), muscle inflammation (e.g., myositis, etc.), brain inflammation (e.g., encephalitis, major depression, etc.), neuroinflammation (e.g., neuritis of the eyes, ears, etc., complex regional pain syndrome, Guillain-Barré syndrome, etc.), eye inflammation (e.g., sty, uveitis, conjunctivitis, etc.), ear inflammation (e.g., otitis media, mastoiditis, etc.), oral inflammation (e.g., stomatitis, periodontitis, gingivitis, etc.), systemic inflammation (e.g., systemic inflammatory response syndrome (sepsis), metabolic syndrome-related diseases, etc.), peritonitis, reperfusion injury, transplant rejection and hypersensitivity reaction, but not limited to these.

The anti-TIGIT antibodies, antigen-binding fragments thereof and/or pharmaceutical compositions comprising the same provided herein can be administered to any animal or cell, for example, an animal selected from mammals, including primates such as humans and monkeys, rodents such as rats, mice, etc., or cells, tissues, body fluids (such as serum) derived from (isolated from) animals, or cultures thereof, for example, cells, tissues, body fluids (serum) isolated from humans.

In addition to the anti-TIGIT antibody or its antigen-binding fragment as an active ingredient, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are commonly used in the preparation of protein drugs, and may be selected from lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil at least one, but not limited thereto. The pharmaceutical composition may also include at least one selected from diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the preparation of pharmaceutical compositions.

Anti-TIGIT antibodies, antigen-binding fragments thereof and/or pharmaceutical compositions can be administered orally or parenterally. For parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be used. Since proteins or peptides are digested during oral administration, the active ingredients in the oral composition can be coated or formulated to prevent digestion in the stomach.

In addition, the anti-TIGIT antibody, its antigen-binding fragment and/or pharmaceutical composition can be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or can be formulated into an extract, powder, powder, granule, tablet or capsule, injection, etc. The composition may also contain a dispersant or a formulation stabilizer.

The content of the anti-TIGIT antibody or its antigen-binding fragment in the pharmaceutical composition or its dosage can be determined according to various factors, such as the formulation method, the administration method, the patient's age, weight, gender, pathology, diet and administration time, administration interval, administration route, excretion rate, reaction sensitivity, etc. The pharmaceutical composition can be based on the active ingredient (anti-TIGIT antibody or antigen-binding fragment) in an amount of 0.00001 mg/kg to 1000 mg/kg, 0.00001 mg/kg to 500 mg/kg, 0.00001 mg/kg to 100 mg/kg, 0.00001 mg/kg to 50 mg/kg, 0.0001 mg/kg to 1000 mg/kg, 0.0001 mg/kg to 500 mg/kg, 0.0001 mg/kg to 1000 mg/kg, 0.0001 mg/kg to 500 mg/kg, 0.0001 mg/kg to 1000 mg/kg, 0.0001 mg/kg to 500 mg/kg, 0.001 mg/kg to 1000 mg/kg. The daily dose of 0.1mg/kg to 500mg/kg, 0.001mg/kg to 100mg/kg, 0.001mg/kg to 50mg/kg, 0.01mg/kg to 1000mg/kg, 0.01mg/kg to 500mg/kg, 0.01mg/kg to 100mg/kg, 0.01mg/kg to 50mg/kg, 0.1mg/kg to 1000mg/kg, 0.1mg/kg to 500mg/kg, 0.1mg/kg to 100mg/kg or 0.1mg/kg to 50mg/kg is used, but is not limited thereto. Daily dose can be formulated into a preparation of unit dosage form, formulated into appropriate parts, or prepared by being placed in a multidose container. In addition, in this specification, drug effective amount refers to the amount of the active ingredient that can show the desired pharmacological activity of active ingredient, and can be within the above dosage range.

Construction of polynucleotides and expression vectors and production of antibodies

In another aspect, a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region or a heavy chain of an anti-TIGIT antibody is provided.

In another aspect, a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or a light chain of an anti-TIGIT antibody is provided.

On the other hand, a recombinant vector is provided, which carries a combination of a nucleic acid molecule encoding a heavy chain complementary determining region, a heavy chain variable region or a heavy chain of an anti-TIGIT antibody and a nucleic acid molecule encoding a light chain complementary determining region, a light chain variable region or a light chain of an anti-TIGIT antibody, or a separated recombinant vector, which carries a nucleic acid molecule encoding a heavy chain complementary determining region, a heavy chain variable region or a heavy chain of an anti-TIGIT antibody and a nucleic acid molecule encoding a light chain complementary determining region, a light chain variable region or a light chain of an anti-TIGIT antibody, respectively. The recombinant vector can be an expression vector for expressing a nucleic acid molecule.

Another aspect provides a recombinant cell harboring a recombinant vector.

As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, such as a plasmid vector, a cosmid vector, and a viral vector, such as a phage vector, a lentiviral vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. The recombinant vector can be constructed by operating a plasmid commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λδZ1, M13, etc.) or a viral vector (e.g., SV40, etc.).

In a recombinant vector, a nucleic acid molecule can be operably linked to a promoter. The term "operably linked" refers to a functional connection between a nucleotide sequence of interest and an expression control sequence (e.g., a promoter sequence). When "operably linked," a regulatory element can control the transcription and/or translation of a polynucleotide of interest.

Recombinant vectors can be constructed as cloning vectors or expression vectors. For recombinant expression vectors, vectors commonly available in the relevant art for expressing foreign proteins in plants, animals or microbial cells can be used. Various methods well known in the art can be used to construct recombinant vectors.

In order to use in a host such as a prokaryotic or eukaryotic cell, a recombinant vector can be constructed accordingly. For example, when a vector is constructed as an expression vector for a prokaryotic host, the vector generally includes a strong promoter for transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. On the other hand, an expression vector for a eukaryotic host includes an operable replication origin in a eukaryotic cell, such as f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin, but is not limited thereto. In addition, the expression vector generally includes a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence.

Recombinant cells can be prepared by importing the recombinant vector into a suitable host cell. As long as it allows the recombinant vector to be cloned and expressed in a stable manner, any host cell known in the art can be used for the present disclosure. Examples of prokaryotic host cells that can be used for the present invention can be selected from Escherichia coli, such as Escherichia coli JM109, Escherichia coli BL21, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X 1776, Escherichia coli W3110, Bacillus, such as Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas. Eukaryotic host cells that can be used for transformation can be selected from, but are not limited to, Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, such as Sp2/0, CHO (Chinese Hamster Ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK, etc.

The nucleic acid molecule or the recombinant vector carrying the nucleic acid molecule can be delivered (introduced) into the host cell using methods known in the relevant art. For example, when the host cell is a prokaryotic cell, _CaCl2 or electroporation methods can be used for such delivery. For eukaryotic host cells, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection or particle bombardment can be used but are not limited to achieve gene introduction.

According to methods well known in the art, in order to select transformed host cells, the phenotype associated with the selection marker can be utilized. For example, when the selection marker is a gene that confers resistance to a certain antibiotic, the host cells can be grown in a culture medium in the presence of the antibiotic to select the transformant of interest.

On the other hand, a method for producing an anti-TIGIT antibody or an antigen-binding fragment thereof is provided, the method comprising the step of expressing a nucleic acid molecule in a host cell. The step of expressing a nucleic acid molecule in a host cell may be a step of culturing a cell anchoring the nucleic acid molecule or a recombinant vector carrying the nucleic acid molecule. The method may also include a step of separating and/or purifying the antibody or antigen-binding fragment from the culture medium after the culturing step.

Beneficial Effects

The anti-TIGIT antibodies or antigen-binding fragments thereof provided herein have the function of blocking the action of TIGIT to activate immunity (e.g., enhancing effector T cell function, regulating Treg activity, increasing cytokine secretion, etc.), and therefore may find advantageous applications as various immune activators and/or immunotherapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing flow cytometry results of an anti-TIGIT antibody (7A6) according to an embodiment.

FIG. 2 shows a sequence alignment of chimeric and humanized antibodies according to an embodiment.

FIG3 a shows the results of epitope mapping of an anti-TIGIT antibody according to an embodiment, and FIG3 b shows the location of the epitope on the tertiary structure of the TIGIT protein.

FIG4 is a graph showing the binding affinity of anti-TIGIT antibodies according to the embodiments to TIGIT in human primary T cells.

FIG. 5 is a graph showing the TIGIT-PVR blocking effect of an anti-TIGIT antibody according to an embodiment.

6 is a graph showing the effects of an anti-TIGIT antibody according to an embodiment on cytokine (IL-2 and IFN-γ) production/secretion in human peripheral blood mononuclear cells (PBMCs).

7a and 7b are graphs showing the cytokine production/secretion effects of anti-TIGIT antibodies according to the embodiments in T cells, and CD4+ T cells (7a) and CD8+ T cells (7b) were analyzed.

Figures 8a to 8d are graphs showing the cytokine production/secretion effect of anti-TIGIT antibodies in T cells according to the embodiment, and the IL-2 concentration in CD4+T cells (8a), the IFN-γ concentration in CD4+T cells (8b), the IL-2 concentration in CD8+T cells (8c), and the IFN-γ concentration in CD8+T cells (8d) were analyzed.

Figures 9a to 9d are graphs showing the cytokine production/secretion effect of the anti-TIGIT antibody according to the embodiment in T cells according to its concentration, and the IL-2 concentration in CD4+T cells (9a), the IFN-γ concentration in CD4+T cells (9b), the IL-2 concentration in CD8+T cells (9c) and the IFN-γ concentration in CD8+T cells (9d) were analyzed.

10a and 10b are graphs showing the effects of anti-TIGIT antibodies according to embodiments on T cell proliferation in human PBMCs, analyzed by CFSE assay (10a) and by Ki67 assay (10b).

FIG. 11 is a graph showing the effect of an anti-TIGIT antibody according to an embodiment on T cell proliferation in the presence of Treg cells.

12 is a graph showing the combined blocking effect of a combination of an anti-TIGIT antibody and an anti-PD1 antibody on TIGIT-PVR/PD-1-PD-L1 according to an embodiment, as analyzed by a cell-based NFAT reporter reaction bioassay.

13a and 13b are graphs showing comparison of blocking effects against TIGIT-PVR/PD-1-PD-L1 between anti-TIGIT antibodies according to the embodiments and reference antibodies when used in combination with anti-PD1 antibodies pembrolizumab (13a) and nivolumab (13b).

14a and 14b are graphs showing the results of cytokine production by human T cells when an anti-TIGIT antibody according to an embodiment is used alone or in combination with an anti-PD1 antibody, and CD4+ cells (14a) and CD8+ cells (14b) were analyzed.

FIG. 15 is a graph showing the expression level of TIGIT ligand CD155 in A375 and SK-OV3 tumor cell lines.

16 is a graph showing the cytotoxicity (cell death rate) of anti-TIGIT antibodies according to embodiments against A375 tumor (melanoma) cell line.

17a and 17b are graphs showing the cytotoxicity (cell death rate) of anti-TIGIT antibodies according to embodiments against the SKOV-3 tumor (ovarian cancer) cell line.

18 is a graph showing the cytotoxicity (cell death rate) of anti-TIGIT antibodies according to embodiments against the SKOV-3 tumor (ovarian cancer) cell line.

19 is a graph showing the cytotoxicity (cell death rate) of an anti-TIGIT antibody according to an embodiment against SKOV-3 tumor (ovarian cancer) cell line when co-cultured with NK cells.

FIG. 20 is a graph showing the in vivo anti-tumor effect of an anti-TIGIT antibody on colon cancer according to an embodiment.

21 is a graph showing the in vivo anti-tumor effect on colon cancer when an anti-TIGIT antibody according to an embodiment is used alone and in combination with an anti-PD1 antibody.

FIG22 shows the results of determining the effect on immune cells in the tumor microenvironment (TME) when an anti-TIGIT antibody according to an embodiment is used in combination with an anti-PD1 antibody.

23 is a graph showing the in vivo anti-tumor effect of an anti-TIGIT antibody according to an embodiment on xenograft tumors derived from liver cancer patients.

FIG. 24 is a graph showing TIGIT expression levels on T cell subsets in the tumor microenvironment (TME).

25 is a graph showing the effect of an anti-TIGIT antibody according to an embodiment on cytokine production in T cells derived from liver cancer patients.

26 is a graph showing the effects of an anti-TIGIT antibody according to an embodiment on cytokine production in T cells derived from lung cancer patients.

27 is a graph showing the effects of an anti-TIGIT antibody according to an embodiment on cytokine production in T cells from colon cancer patients.

28a and 28b are graphs showing the activation (cytokine production) effects of anti-TIGIT-Fab fragments according to the embodiments in central memory T cells (28a) and effector memory T cells (28b).

29 is a graph showing the cytotoxicity (cell death rate) of anti-TIGIT-Fab fragments according to an embodiment against A375 tumor cells.

FIG. 30 is a graph showing the expression levels of TIGIT in T cell subsets.

31a to 31d are graphs showing the remaining cell counts of Tregs, CD4+ T cells, and CD8+ T cells after treatment with an anti-TIGIT antibody according to an embodiment.

FIG. 32 is a graph showing the cell counts of Treg and NK cells after treatment with an anti-TIGIT antibody according to an embodiment.

FIG. 33 is a graph showing the residual cell count of Treg cells after treatment with an anti-TIGIT antibody according to an embodiment in the presence of NK.

34a and 34b are graphs illustrating the influence of cytokine production in T cells depending on whether the anti-TIGIT antibody according to the embodiment blocks FcgRIIIA (34a: CD4+ T cells, 34b: CD8+ T cells).

Mode for Carrying Out the Invention

The present invention can be better understood by the following examples, which are intended to illustrate the present invention, rather than to limit the present invention. For those skilled in the art, it is obvious that the following examples can be modified without departing from the spirit of the present invention.

Example 1: Construction of anti-TIGIT antibodies

1.1. Preparation of anti-TIGIT monoclonal antibodies

Five BALB/c mice were immunized by cross-injection of an MMB-designed immunogen (Ag1585_IMM) and human TIGIT protein (aa22-138; SinoBiologics, UniProtKB/SwissProt Q495A1-1) five times over 19 days. Lymphocytes were collected from five mice, combined, purified, and then fused with SP2/0 myeloma cells. The fused cells were propagated in HAT selective single-step cloning medium, and the resulting 1896 hybridoma clones were transferred and cultured in 96-well plates.

The peptide Ag1585_IMM used as an immunogen is modeled to reflect the relationship based on folding and proximity within the parent protein, and by maximizing the potential of the immunogenic protein, it is expected to be used to produce antibodies with full activity against the corresponding epitope within the whole protein. The synthesized peptide Ag1585_IMM includes 20 amino acids from the 51st amino acid residue (T) to the 70th amino acid residue (N) of human TIGIT. Table 3 below summarizes the information about the peptide Ag1585_IMM:

Table 3

Table 3 above illustrates the structural features of the modeled Ag1585_IMM and its alignment with the TIGIT structure (PDB: 5V52). Low RMSD scores indicate good alignment. Peptides were synthesized based on the corresponding immunogen sequences.

Using indirect ELISA, the hybridoma tissue culture supernatant was screened for the effects of the immune antigen Ag1585_IMM and recombinant human TIGIT (His). For effective clones (positive clones), further indirect ELISA tests were performed on the screening antigen (recombinant human TIGIT Fc chimeric protein (R&D systems, 9464-TG)) and cells to confirm Ig secretion and specificity. CHO-K1 (ATCC, CCL-61) cell line was used as a negative control.

ELISA was performed under the following conditions:

- ELISA plates were coated O/N at 4°C with 100 μL/well of carbonate coating buffer (pH 9.6) containing recombinant human TIGIT (rhTIGIT) protein at a concentration of 0.1 μg/well.

- Block the plates with 3% nonfat dry milk in PBS for 1 hour at room temperature.

- Incubate with goat anti-mouse secondary antibody IgG/M(H+L)-HRP in PBS-Tween (1:10000) at 37°C with shaking for 1 hour (100 uL/well).

- All washing steps were performed with PBS-Tween for 3 minutes.

- Add TMB substrate at a concentration of 50 uL/well and incubate in the dark for 5 to 10 minutes, then stop the reaction with an equal volume of 1 M HCl.

By isotyping, IgM-expressing clones are identified and eliminated, and IgG-expressing clones are collected.

The collected positive clones were subcloned to identify the clones with stable expression. The culture supernatant containing the selected clones was eluted through a protein A column, and the buffer was exchanged into PBS. The purified antibodies were tested by indirect ELISA against the screening antigen (recombinant human TIGIT (T103) Fc chimeric protein; R&D Systems) and the negative control antigen (His peptide).

Binding of the selected lead antibody clone (7A6) was measured by flow cytometry analysis. CHO-K1 (ATCC, CCL-61) and CHO-K1 expressing human TIGIT (CHO-K1TIGIT) (Genscript, M00542) cells were trypsinized, counted, resuspended at a concentration of 2x10 ⁶ cells/ml, and incubated with Fc block (BD Bioscience 564220) for 30 minutes. After incubation, cells were added to 96-well plates and the added antibodies (single point) were serially diluted (8 points, starting from 5μg/ml, diluted 3 times). For CHO-K1 cells (negative control), they were tested at a single concentration of 5μg/ml, which was equivalent to the highest concentration titrated on CHO-K1 TIGIT cells. After 30 minutes of incubation, antibody binding was measured using 4μg/ml (protected from light) of anti-mouse IgG (H+L) Alexa Fluor 647 antibody (Sigma A21236). Flow cytometry was performed using an AccuriC6 flow cytometer, and the data were analyzed using FlowJo. The results obtained are shown in Figure 1.

The hybridoma cell pellet corresponding to the stable expression clone was dissolved, mRNA was extracted, and the variable region DNA of the heavy chain and light chain was cloned into a sequencing vector. The DNA sequence of the heavy chain and light chain was analyzed. The sequence analysis results of the mouse anti-TIGIT clone (7A6) are listed in Table 4 below.

Table 4

(In Table 4, the underlined regions represent CDR1, CDR2, and CDR3 of the heavy chain and light chain, respectively)

1.2. Humanization of mouse anti-TIGIT clones

To confirm the amino acid residues that are critical for antibody structure and binding, protein structure models of monoclonal antibody variable regions (mAb V regions) were analyzed. This information was used in conjunction with computer designs of human antibody structures. A large number of preliminary sequence fragments were screened for potential use in 7A6 humanization and tested for peptide binding to human MHC class II alleles. Where possible, sequence fragments that were identified as significant non-human germline binders to human MHC class II were discarded. This reduced the set of fragments, which were reanalyzed using the methods described above to ensure that the junctions between fragments did not include potential T-cell epitopes. To generate complete variable regions (V regions) containing no or reduced significant T-cell epitopes, sequence fragments were assembled to design humanized variable regions that either circumvented potential T-cell epitope recognition or contained minimized T-cell epitopes (deimmunization).

For gene synthesis and expression in mammalian cells, five heavy chain (VH1 to VH5) and five light chain (Vκ1 to Vκ5) sequences were selected.

A summary of stable IgG antibodies produced by transient transfection, including one chimeric (VH0/Vκ0) and 25 humanized variants (marked as ‘○’), is shown in Table 5 below:

Table 5

(In Table 5, the given values (%) represent the percentage of humanization (determined as the percentage of homology to the closest matching human germline)).

The chimeric antibody (VH0xVκ0) and humanized antibody sequences derived by applying the combination of the 7A6 clones in Table 4 to the combination in Table 5 are given in Tables 6 and 7 and Figure 2. In Tables 6 and 7 and Figure 2, the amino acid sequence numbers and CDR regions are determined according to the Kabat definition, and the amino acid residues that differ from the CDR and parental sequences (VH0 or Vκ0) determined above are shaded in Figure 2.

Table 6

Heavy chain (constant region: human IgG1)

Table 7

Light chain (constant region: kappa (denoted as K, k, or κ))

Among the antibodies generated, the 7A6 VH3/Vk5 hIgG1 antibody underwent the following point mutations in the Fc region of its heavy chain to generate an Fc engineered variant:

(1) S240D, A331L, and I333E (DLE variants): 7A6 VH3/Vk5-DLE;

(2)N298A: 7A6 VH3/Vk5 N298A;

(3) S299A, E3334A and K335A (AAA variant): 7A6 VH3/Vk5 AAA; or

(4) L235A, L236A and P330G (LALAPG variants): 7A6 VH3/Vk5 LALAPG

Table 8 lists the sequences of the heavy and light chains of antibodies containing the above-mentioned Fc engineered variants.

Table 8

(In Table 8, the variable region is underlined and the mutated amino acid residues in the Fc region are marked in bold)

1.3. Transient expression of chimeric and humanized IgG1 antibodies

Using the PEI transfection method, the coding DNA of the VH0/Vκ0 chimeric antibody and the coding DNA of the humanized heavy chain and light chain combination (a total of 25 humanized pairs) were transiently transfected into HEK293 EBNA adherent cells (LGCStandards, Teddington, UK) in 6-well plates. After transfection, the cells were cultured for 7 days. Samples were collected and the antibody concentration was measured on Octet QK384 using a protein A biosensor (MolecuLar Devices, Wokingham, Berkshire, UK) based on human IgG1 antibody standards.

The results obtained are listed in Table 9 below:

Table 9

Table 9 shows the IgG concentration (μg/ml) in the supernatant after various combinations of chimeric antibodies (VH0/Vκ0) and humanized antibodies were expressed in HEK cells.

As shown in Table 9, all antibodies tested were well expressed, and in particular, except for VH5Vκ1 and VH5Vκ5, all humanized variants were better expressed than the VH0/Vk0 chimeric antibody.

1.4. Single-cycle kinetic analysis of chimeric and humanized variants

To evaluate the binding of all variants to the human TIGIT antigen and select the humanized IgG antibody with affinity closest to the chimeric antibody (VH0Vκ0), a single cycle kinetic analysis (cartoon) was performed in the supernatant from the transfected cell culture. Kinetic tests were performed at 25°C using a Biacore T200 running Biacore T200 control software V2.0.1 and evaluation software V3.0 (GE Healthcare, Uppsala, Sweden).

In order to reduce non-specific binding to the reference surface, HBS-P+ (GE Healthcare, Uppsala, Sweden) supplemented with 1% BSA w/v (Sigma, Dorset, UK) was used as a flow buffer and was also used for dilution of ligands and analytes. The supernatant containing IgG was diluted to 1 μg/ml in running buffer. At the beginning of each cycle, the antibodies were loaded onto Fc2, Fc3 and Fc4 on an anti-human sensor chip (GE Healthcare, Little Chalfont, UK). The IgG antibody was captured at a flow rate of 10 μL/min to achieve an immobilization level (RL) of about 208RU. Then, the surface was allowed to stabilize.

To minimize potential mass transfer effects, recombinant human TIGIT (from Sino Biological) was used as the analyte and injected at a flow rate of 40 μL/min to obtain single-cycle kinetic data. Antigen was diluted in flow buffer to a concentration range of 1.25 nM to 10 nM, diluted 2-fold (at 4 points), and used without regeneration between concentrations. For each of the four increasing antigen concentrations, the binding phase was monitored for 210 seconds, and a single dissociation phase was measured for 900 seconds after the last antigen injection. The sensor chip surface was regenerated by a single injection of 3M MgCl ₂ .

The dual reference sensorgrams were fitted with the Langmuir (1:1) binding model, and the fit of the data to the model was evaluated using chi-square values, which describe the deviation between the experimentally obtained curve and the fitted curve (observed and predicted). The fitting algorithm aims to minimize the chi-square value. The kinetic constants determined by the 1:1 model fitting curve are shown in Table 10.

Table 10

Table 10 shows the single cycle kinetic parameters for binding of chimeric antibodies (VH0/Vκ0) and humanized variants to human TIGIT antigen measured using Biacore T200. Relative _KD was calculated by dividing the _KD of the humanized variants by the KD of the VH0/Vκ0 chimeric antibody analyzed in the same experiment.

As shown in Table 10, it was found that all tested humanized variants (antibodies) had K _values 2.55 times higher than VH0/Vk0 for binding to human TIGIT. Considering the relative expression level, percentage of humanization and relative K _values (obtained from Biacore single cycle kinetic analysis of supernatants), six humanized variants (VH2/Vκ5, VH3/Vκ4, VH3/Vκ5, VH4/Vκ4, VH4/Vκ5 and VH5/Vκ5) were selected for subsequent thermal stability analysis.

1.5. Thermal stability evaluation

To obtain information about the thermal stability of an antibody from the temperature at which it transitions from its native state to its denatured state (unfolding), thermal ramp stability experiments (Tm and Tagg) were performed. This unfolding process occurs over a narrow temperature range, and the midpoint of this transition is called the "melting temperature" or "Tm." Because the protein undergoes a conformational change at this point, the fluorescence of the Sypro Range (which binds to exposed hydrophobic regions of the protein) is measured to determine the melting temperature of the protein.

Samples were diluted in PBS to a final test concentration of 0.5 mg/ml, and Sypro ^TM Orange (160× stock solution; Sigma-Aldrich) was added to a final concentration of 20×. Each sample mixture was loaded into a UNi microcuvette twice, 9 μL each time. The samples were thermally ramped from 15°C to 95°C (at a ramp rate of 0.3°C/min, with an excitation wavelength of 473 nm). The complete emission spectrum from 250nm to 720nm was measured, and the inflection point (Tonset and Tm) of the transition curve was calculated using the area under the curve from 510nm to 680nm. Static light scattering (SLS) at 473nm was monitored to detect protein aggregation, and Tagg (aggregation start) was calculated from the resulting SLS curve. Data analysis was performed using UNcle ^TM software (ABZENA) version 4.0.

The results obtained are summarized in Table 11 below:

Table 11

The thermal stability values obtained using the UNcle biostability platform are shown in Table 11. As shown in Table 11, all humanized antibodies tested exhibited similar thermal stability levels as the chimeric antibodies.

1.6. Affinity Measurement of Humanized Variants Using Multi-Cycle Kinetic Analysis

To measure the binding affinity of chimeric antibodies and six major humanized variants (VH2/Vκ5, VH3/Vκ4, VH3/Vκ5, VH4/Vκ4, VH4/Vκ5 and VH5/Vκ5) to human TIGIT antigen, multi-cycle kinetic analysis was performed on the purified proteins (antibodies). Kinetic experiments were performed at 25°C using a Biacore T200 operated using control software V2.0.1 and evaluation software V3.0 (GE Healthcare, Uppsala, Sweden).

HBS-P+ (GE Healthcare, Uppsala, Sweden) supplemented with 1% BSA w/v (Sigma, Dorset, UK) was used as a flow buffer and also for dilution of ligands and analytes. The purified lead antibody was diluted to a final concentration of 1 μg/ml in the flow buffer and loaded onto the Fc2, Fc3, and Fc4 sites on the anti-human IgG CM5 sensor chip (GE Healthcare, Little Chalfont, UK) at the beginning of each cycle. The antibody was captured at a flow rate of 10 μL/min until a response level (RL) of approximately 150 RU was reached, followed by a stabilization period.

To minimize potential mass transfer effects, recombinant human TIGIT (Acrobiosystems, China) was introduced as the analyte at a flow rate of 50 μL/min to obtain multi-cycle kinetic data. The antigen (TIGIT) was diluted two-fold in the flow buffer from 0.406 nM to 30 nM (7 points), and each concentration was applied without regeneration between cycles. For each concentration, the binding phase was monitored for 240 seconds, followed by the dissociation phase for 900 seconds. The sensor chip surface was regenerated between kinetic cycles by injecting 3M MgCl ₂ twice. To ensure the stability of the surface and the analyte during the kinetic cycles, repeated injections of blanks and single concentrations of analytes were incorporated into the kinetic runs.

The results obtained from the above experiments, in particular the results for the chimeric antibody (VH0/Vκ0) and the VH3/Vκ5 antibody, are summarized and presented in Table 12:

Table 12

Antibody Analytes ka(1/Ms) Kd(1/s) KD(M) RMAX Chi2(RU2) VH0/Vk0 hTIGIT 2.28E+06 1.76E-04 7.70E-11 28.2 0.425 VH3/Vk5 hTIGIT 2.04E+06 1.77E-04 8.67E-11 26 0.246

Table 12 shows the kinetic constants determined by the 1:1 model fitting curve. As shown in Table 12, the binding affinity (KD) of human TIGIT protein is 77 pM for antibody 7A6 VH0Vk0 and 86.7 pM for antibody VH3/Vk5, which are similar.

1.7. Epitope mapping

The epitopes of the antibodies were identified by peptide mass fingerprinting (PMF) and H/D exchange (hydrogen/deuterium exchange).

To detect the incorporation of deuterium atoms in TIGIT and the mixture of TIGIT and antibody 7A6 Vh3/Vk5, the peptide mass fingerprint (PMF) of the samples was optimized. Proteolytic digestion of protein samples was performed under quenching conditions to limit the back exchange of deuterium atoms that may occur during protein digestion and chromatography.

TIGIT solution (SEQ ID NO: 30; NCBI reference sequence NP_776160.2; UniProtKB/SwissProt Q495A1-1) (15 μM, 150 μL) and a mixture of TIGIT and Vh3Vk5, the ratio of TIGIT _: Vh3Vk5 = (15 μm:30 μm, 150 μL) were prepared. A control experiment was carried out by mixing 4 μL of the above protein sample with 56 μL of labeling buffer (5 mM _K2HPO4 ; 5 mM _KH2PO4 , _D2OpH 6.6) _and 56 _μL of equilibration buffer (5 mM _K2HPO4 ; 5 _{mM KH2PO4} , pH 7.0). 15× dilution solution (60 μL, TIGIT: Vh3Vk5; 1 μM: 2 μM) was incubated for 15 seconds, 60 seconds, 180 seconds, 600 seconds, 1800 seconds and 7200 seconds, respectively, after which 50 μL of quenching solution (50 mM K ₂ HPO ₄ ; 50 mM KH ₂ PO ₄ ; GuCl 2.0 M, TCEP 200 mM, pH 2.3, 0°C) was added to the protein sample and incubated for another 20 seconds. Immediately after incubation, 80 μL of the quenched protein solution was injected into the proteolytic pepsin column and kept at 15°C for 5 minutes. After protein digestion, the generated pepsin peptides were analyzed using liquid chromatography with C18 chromatography (HDX Manager Waters) before MSe Xevo-G2-XS analysis. These tests were performed three times.

H/D exchanged peptides were analyzed using DynamX 3.0 software. Deuterium levels were determined by considering the average of all results with high and medium confidence. Deuteration levels were calculated based on the centroid of the experimental isotope cluster.

From the results of HDX-MS analysis, significant differences in deuterium incorporation within TIGIT were observed when incubated alone or with 7A6 VH3/Vk5. The major differences in deuterium incorporation were observed in amino acid residues 51 to 70 (TAQVTQVNWEQQDQLLAICN; SEQ ID NO: 31), which are responsible for the epitope region of 7A6 Vh3Vk5.

The results are shown in Figures 3a and 3b. Figure 3a shows the results of HDX-MS analysis (with high levels of deuterium incorporation at sites 51 to 70 of TIGIT (TAQVTQVNWEQQDQLLAICN; SEQ ID NO: 31)). Figure 3b schematically shows the ribbon structure of TIGIT; the top is a top view, the bottom is a side view, and the epitope sites are indicated by arrows.

1.8. Transient Antibody Expression and Purification of Lead Humanized Antibodies

The heavy and light chain variable region sequences of 7A6 VH3/Vk5 were synthesized, as well as restriction enzyme sites for cloning vector construction. The synthetic sequences of the heavy and light chains (heavy chain: Mlu I and Sal I (New England Biolabs); light chain: BssH II and BamH I (New England Biolabs)) were treated with appropriate restriction endonucleases and ligated into an expression vector containing a human IgG1 constant region treated with the same restriction endonucleases. The heavy and light chain cDNA constructs were sequenced. Giga prep was performed to prepare DNA for transient transfection in CHO cells. Giga prep was performed using the PureLink ^TM HiPure Expi PlasmidGigaprep Kit (Thermo Fisher Scientific, K210009XP). This Gigaprep kit is designed to amplify plasmid DNA in Escherichia coli and then purify it using anion exchange chromatography. CHO cells (Evitria) were cultured using animal component-free and serum-free medium (CD CHO medium, Thermofisher). The produced antibodies were purified from the culture supernatant by protein A purification method using MabSelect ^™ SuRe ^™ . Sequential purifications were performed using size exclusion chromatography (SEC) to obtain a purity greater than 95%. The purified antibodies were characterized by measuring the absorbance at 280 nm and by SDS-PAGE.

1.9. Specificity and binding affinity of TIGIT in primary human T cells

Anti-TIGIT 7A6VH3/Vk5 antibody was conjugated to Alexa Fluor using the APEX ^™ Antibody Labeling Kit (Invitrogen) according to the manufacturer’s instructions. (AF488) conjugated. T cells were isolated from human PBMC using microbeads (EasySep ^™ ) and labeled anti-TIGIT antibodies (anti-TIGIT 7A6 VH3/Vk5 antibodies; 50ng/ml, 250ng/ml, 750ng/ml, 1250ng/ml and 2500ng/ml) at different concentrations. Binding was measured using a flow cytometer (CytoFLEX, Beckman CouLter) and the data obtained were analyzed using FlowJo software (TreeStar, Inc). The binding of anti-TIGIT antibodies was confirmed on CD3+, CD4+ and CD8+ T cells.

The results obtained are shown in Figure 4. As shown in Figure 4, the tested anti-TIGIT antibodies bound to primary T cells, and the binding to the TIGIT molecule (per cell) increased as the concentration of the anti-TIGIT antibody increased.

Reference Example: Preparation of reference antibodies

The reference antibodies used as references in the following examples were constructed based on the sequence information disclosed in the corresponding patents or purchased from manufacturers. The corresponding patents or manufacturers of each antibody are summarized as follows:

22G2(BMS): US2016/0176963 A1,

31C6(Merck)：WO2016/028656 A1，

4.1D3 (Genentech): WO2017/053748 A2,

TIG1(Arcus)：WO2017/152088 A1，

313M32(Mereo)：US2016/0376365 A1，

Tirelizumab (Roche): purchased from CrownBio,

10A7 (Genentech): purchased from Creative Biolabs,

MBSA43: purchased from eBioscience,

Pembrolizumab and nivolumab: purchased from InvivoGen.

Example 2: Measuring the biological activity of anti-TIGIT antibodies by cell-based reporter assay

The blocking effect of anti-TIGIT antibodies on the interaction between TIGIT and its receptor, the poliovirus receptor (PVR) (TIGIT-PVR blocking effect) was analyzed using a cell-based NFAT reporter reaction bioassay (Promega). TIGIT effector cells (Promega) were added to cell assay buffer (90% RPMI 1640/10% FBS), and the cell suspension containing TIGIT effector cells was incubated at 37°C for 16 hours. Anti-TIGIT antibodies (7A6VH3/Vk5) or reference antibodies were prepared in PBS buffer and added to the pre-incubated cell suspension. CD155 aAPC/CHO-K1 cells (Promega) were added to the mixture containing cells and antibodies and incubated at 37°C for 6 hours. Promega ^TM Luminescence was measured using a plate reader. Curve fitting software (GraphPad _EC50 values of antibody responses were measured using the ELISA software.

The results obtained are shown in Figure 5. As shown in Figure 5, the activation signal of T cells increased due to TIGIT-PVR blockade by anti-TIGIT 7A6 VH3/Vk5 antibodies. Compared with reference antibodies such as BMS (22G2), Arcus (TIG1), Genentech (4.1D3) and Mereo (313M32), the anti-TIGIT 7A6 VH3/Vk5 antibodies of the present application showed significantly higher effects (significantly lower EC ₅₀ values).

Example 3: Cytokine production effects of anti-TIGIT antibodies

3.1. Preparation of human peripheral blood mononuclear cells and tumor infiltrating lymphocytes

Peripheral blood mononuclear cells (PBMC) were obtained from adults (whole blood leukocyte cones, NHS Blood and Transplant, UK) or purchased from STEMCELL Technologies. After appropriate ethical review and prior consent from the "Liver and Gastrointestinal Research Center, University of Birmingham, UK", patients with hepatocellular carcinoma (HCC) and healthy normal donors were tested in this working example. PBMC and liver-derived lymphocytes were prepared from liver tissue of HCC patients. Liver-infiltrating lymphocytes were isolated from 0.5cm to 1cm liver puncture biopsy. Subsequently, the tissue was homogenized in 2mL to 3mL Dulbecco's phosphate buffered saline (GIBCO) using a Dounce tissue grinder. PBMCs from patients with melanoma, ovarian cancer, CRC (colorectal cancer), HCC, NSCLC (non-small cell lung cancer) and pancreatic cancer were obtained from Cureline, Inc. The liver-infiltrating lymphocytes and PBMCs obtained from cancer patients were then analyzed as follows.

3.2. Determination of cytokine production in human PBMCs

3.2.1. Anti-TIGIT antibodies increase cytokine secretion in human PBMCs

PBMCs were cultured with anti-CD3 antibodies in the presence of anti-TIGIT 7A6 VH3/Vk5 antibodies (2 μg/ml and 10 μg/ml). The group not treated with anti-TIGIT antibodies was used as a control. After culture, the samples were centrifuged at 400 x g for 10 minutes, and the supernatant was used for multiple immunoassays. Cytokine concentrations were measured using the Bio-Plex cytokine assay (human cytokine assay, including IFN-γ, IL-2). All analyses were performed according to the manufacturer's instructions and the results were read on a Bio-Plex 200 array reader (Bio-Rad).

The results obtained (IFN-γ and IL-2 concentrations) are shown in Figure 6. As shown in Figure 6, anti-TIGIT 7A6 VH3/Vk5 antibody significantly induced the secretion of Th1/Tc1 cytokines in human PBMCs. Unless otherwise specified in the specification and drawings, anti-TIGIT 7A6 VH3/Vk5-IgG1 is referred to as anti-TIGIT 7A6 antibody or anti-TIGIT 7A6 VH3/Vk5 antibody.

3.2.2. Anti-TIGIT antibodies increase cytokine production in human T cells

Cytokine production was measured in human T cells (CD4+T cells and CD8+T cells). Cells (500,000 cells/reaction) were cultured with anti-CD3 monoclonal antibodies (BD Biosciences, catalog number 555336; 0.2 μg/ml) and anti-CD28 monoclonal antibodies (BD Biosciences, catalog number 555725; 1 μg/ml) in the presence of anti-TIGIT 7A6VH3/Vk5 antibodies. After culture, cells were centrifuged at 400xg for 10 minutes at 4°C, and cytokine concentrations within T cells were measured. In this regard, intracellular cytokines were stained as follows: cells were stained with Zombie Aqua fixable dead cell dye solution (Biolegend) and labeled on ice for 30 minutes with fluorophore-conjugated antibodies to CD4+ and CD8+T cell surface markers. The anti-CD3, anti-CD4 and anti-CD8 antibodies (all used for CD4+ and CD8+ T cell surface staining), anti-IL-2, anti-IFNg and anti-TNFa antibodies used were purchased from Biolegend.

Cells were fixed and permeabilized using the eBioscience ^TM Foxp3/Transcription Factor Fixation/Permeabilization Concentration and Dilution Kit (eBiosciences) according to the manufacturer's instructions. Cells were then stained with fluorophore-conjugated antibodies to intracellular IL-2, IFNγ, and TNFα. Cells were analyzed using a flow cytometer (CytoFLEX, Beckman CouLter) and data were analyzed using FlowJo software (BD Biosciences).

The results are shown in Figures 7a (CD4+T cells) and 7b (CD8+T cells). As shown in Figures 7a and 7b, the anti-TIGIT 7A6 VH3/Vk5 antibody of the present application increased the production of Th1/Tc1 cytokines (IL-2, IFN-γ) in T cells, demonstrating its effect in enhancing immune response. The results obtained show that the anti-TIGIT 7A6 VH3/Vk5 antibody has an effective effect of enhancing effector T cell function in a dose-dependent manner.

Comparison of the potency of anti-TIGIT antibodies and reference antibodies

3.3.1. Anti-TIGIT antibodies have enhanced immune enhancement compared to anti-PD1 drugs

The immune effect of anti-TIGIT antibodies on cytokine production was measured using the method described in Example 3.2.2. For comparison, anti-PD1 antibodies, pembrolizumab (denoted as "Pem analog") and nivolumab (denoted as "Niv analog") were used as reference antibodies. After treatment with anti-TIGIT antibodies or anti-PD1 antibodies, analysis was performed by flow cytometry. Both anti-PD-1 antibodies and anti-TIGIT antibodies were used at a concentration of 10 μg/ml.

The results obtained are depicted in Figures 8a (IL-2 concentration in CD4+ T cells), 8b (IFN-γ concentration in CD4+ T cells), 8c (IL-2 concentration in CD8+ T cells), and 8d (IFN-γ concentration in CD8+ T cells). As shown in Figures 8a to 8d, anti-TIGIT 7A6 VH3/Vk5 antibodies induced higher levels of CD4+ and CD8+ T cell activation compared to pembrolizumab or nivolumab.

3.3.2. Anti-TIGIT 7A6 antibody enhances Th1/Tc1 cytokine production compared to reference anti-TIGIT antibody

After culturing T cells with anti-TIGIT antibodies at different concentrations (2 μg/ml, 5 μg/ml, 10 μg/ml), cytokine production was measured using the method described in Example 3.2.2. The reference anti-TIGIT antibodies used for comparison were as described above.

The results obtained are shown in Figure 9a (IL-2 concentration in CD4+T cells), Figure 9b (IFN-γ concentration in CD4+T cells), Figure 9c (IL-2 concentration in CD8+T cells) and Figure 9d (IFN-γ concentration in CD8+T cells). As shown in Figures 9a to 9d, it was observed that the anti-TIGIT 7A6V H3/Vk5 antibody enhanced the immune response by increasing the production of Th1/Tc1IFN-γ and IL-2 cytokines. Compared with the comparative anti-TIGIT antibody, the anti-TIGIT 7A6 VH3/Vk5 antibody showed a stronger increase in cytokine production.

Example 4: Effect of anti-TIGIT antibodies on T cell proliferation

Effect of anti-TIGIT antibodies on T cell proliferation in human PBMCs

10 μg/ml of anti-TIGIT antibody (7A6 VH3/Vk5-IgG1 or 7A6 VH3/Vk5-IgG4) was co-incubated with plate-bound anti-CD3 antibody (BD Biosciences) (0.2 μg/ml), PBMC (500,000 cells/reaction) and Cytostim activator (Miltenyi Biotec). CFSE (carboxyfluorescein succinimidyl ester) was measured on the Live-Cell Analysis System (Satorius), and Ki67 was measured on a flow cytometer.

The results obtained are shown in Figure 10a (CFSE assay results) and Figure 10b (Ki67 assay results) (no stimulation: no Cytostim activator nor antibody treatment; control: no antibody treatment). As shown in Figures 10a and 10b, the anti-TIGIT antibodies 7A6 VH3/Vk5-IgG1 and 7A6 VH3/Vk5-IgG4 of the present application both induced T cell proliferation, indicating that the immune response induced by the anti-TIGIT antibodies can last for a long time.

4.2. Blocking effect of anti-TIGIT antibodies on regulatory T cells inhibiting CD8+ T cell proliferation

CD8+T cells and Treg (regulatory T cells) were isolated using a human CD8+T cell isolation kit (Miltenyi Biotec 130-096-495) and a CD4+CD25+CD127-dim reg T cell isolation kit II (130-094-775). The isolated CD8+T cells were stained with a cell tracker violet proliferation kit (Thermo Fisher) and inoculated to a CD8:Treg ratio of 4:1. The cells were activated with CD3/CD28 Dynabeads (ThermoFisher), and 10 μg/ml of anti-TIGIT 7A6VH3/Vκ5 antibody was added to the appropriate wells. On day 3, the beads were washed and 50 ng/ml of IL-2 was added. On day 7, cells were stained with surface marker antibodies for live/dead (ThermoFisher LIVE/DEAD Fixable Near-IR DEAD Cell Stain Kit), CD3, CD8, and CD4 (all obtained from Biolegend) and analyzed on a flow cytometer (CytoFlex, Beckman Coulter).

The results obtained are shown in Figure 11. As shown in Figure 11, the proliferation of CD8+T cells decreased with the addition of Tregs, but this inhibition of CD8+T cell proliferation was blocked by anti-TIGIT antibodies. This means that anti-TIGIT antibodies blocked Treg-mediated inhibition of CD8+T cell proliferation.

Example 5: Synergistic effect of anti-TIGIT antibody and anti-PD1 drug combination

5.1. Biological Characterization of Anti-TIGIT/Anti-PD1 Antibodies by Cell-Based Reporter Assays

The synergistic effect of the combination of anti-TIGIT antibody and anti-PD antibody (pembrolizumab or nivolumab) was tested. The combined blocking effect of anti-TIGIT antibody and anti-PD antibody on TIGIT-PVR and PD-1-PD-L1 was analyzed using a cell-based NFAT reporter reaction bioassay (Promega). PD-1+TIGIT+ effector cells (Promega; 1×10 ⁵ cells/reaction) were added to cell assay buffer (90% RPMI 1640/10% FBS), and the resulting cell suspension containing PD-1+TIGIT+ effector cells was cultured at 37°C for 16 hours. PD-L1+CD155 aAPC/CHO-K1 cells (Promega; 4×10 ⁴ cells/reaction) were prepared in cell recovery medium (90% Ham's F-12, 10% FBS). A mixture of anti-TIGIT 7A6 VH3/Vk5 antibody and pembrolizumab or nivolumab was added to a cell suspension containing PD-1+TIGIT+ effector cells (1×10 ⁵ /reaction) and PD-L1+CD 155 aAPC/CHO-K1 (4×10 ⁴ /reaction) and cultured at 37°C for 6 hours. As a control, anti-TIGIT antibody or anti-PD1 antibody was administered alone. When used alone, the antibody was administered at a concentration of 0.02048, 0.512, 1.28, 3.2, 8 or 20 (μg/ml). For combined treatment, anti-TIGIT antibody was used at a concentration of 0.02048, 0.512, 1.28, 3.2, 8, or 20 (μg/ml), while the concentration of anti-PD-1 antibody (pembrolizumab or nivolumab) was also 0.02048, 0.512, 1.28, 3.2, 8, or 20 (μg/ml).

After incubation, Bio- ^Glo reagent was added and incubated at ambient temperature for 10 minutes. Luminescence was measured using a plate reader. Curve fitting software (GraphPad _EC50 values of antibody responses were determined using ELISA software.

The results obtained are shown in Figure 12. As shown in Figure 12, it was observed that the combination of anti-TIGIT antibody and pembrolizumab or nivolumab exhibited a synergistic effect.

Comparison of biological activities between anti-PD1/reference antibodies and anti-TIGIT/anti-PD1 antibodies

The combined effect of anti-TIGIT antibody and anti-PD1 antibody blocking TIGIT-PVR/PD-1-PD-L1 was analyzed using a cell-based NFAT reporter reaction bioassay (Promega). Referring to Example 5.1, anti-TIGIT antibody (7A6) or reference anti-TIGIT antibody was co-cultured with pembrolizumab or nivolumab. For comparison, the reference antibody was cultured with the cell mixture and treated in the same manner.

When used alone, the antibodies were used at a concentration of 0.013, 0.032, 0.082, 0.204, 0.512, 1.28, 3.2, 8, or 20 (μg/ml). For combined treatment, the concentration of anti-TIGIT antibody was 0.013, 0.032, 0.082, 0.204, 0.512, 1.28, 3.2, 8, 20 (μg/ml), while the concentration of anti-PD-1 antibody (pembrolizumab or nivolumab) was also 0.013, 0.032, 0.082, 0.204, 0.512, 1.28, 3.2, 8, 20 (μg/ml).

The results obtained are shown in Figure 13a (combined with pembrolizumab) and Figure 13b (combined with nivolumab). As shown in Figures 13a and 13b, the anti-TIGIT antibody combined with pembrolizumab and nivolumab showed a higher enhancement effect compared to the combination of anti-PD1 antibody and reference anti-TIGIT antibody.

5.3. Immune effects of anti-TIGIT/anti-PD1 antibodies on immune T cells

Cytokine production of human T cells was measured during treatment with anti-TIGIT/anti-PD1 antibodies in combination or alone. Anti-TIGIT antibody 7A6 VH3/Vk5 (2 μg/ml) was premixed with pembrolizumab (2 μg/ml or nivolumab (2 μg/ml) and added to cell cultures. Intracellular cytokines were stained with reference to Example 3.2.2.

The results obtained are shown in Figure 14a (CD4+ cells) and Figure 14b (CD8+ cells). It can be seen from the results that when anti-TIGIT 7A6VH3/Vk5 antibody and anti-PD1 antibody are used in combination, the anti-tumor activity based on Tc1/Th1 cytokine production in human primary T cells is enhanced.

Example 6: Analysis of cytotoxicity of anti-TIGIT antibodies on tumor cells

6.1. Expression level of TIGIT ligand PVR (CD155) in tumor cells

Target tumor cells (A375 and SK-OV3 cells) (ATCC) were stained with anti-CD155 PE-Cy7 (Biolegend) at a dilution of 1:25 to assess the expression of CD155 (PVR) and analyzed using flow cytometry.

The results obtained are shown in Figure 15. As shown in Figure 15, A375 and SK-OV3 tumor cells showed significant expression levels of the TIGIT ligand CD155.

6.2. Cytotoxicity of anti-TIGIT antibodies against A375 melanoma cells

Anti-TIGIT antibodies (7A6 VH3/Vk5 and 7A6 VH3/Vk5-DLE) at a concentration of 10 μg/ml were co-cultured with the A375 cell line (melanoma) (2.5×10e4 cells/reaction) in the presence of PBMC (250,000 cells/reaction) and Cytostim (MilteniBiotec) for 72 hours to test the cytotoxicity against A375 tumor cells. For comparison, the anti-TIGIT10A7 reference antibody (Genentech) was used. In addition, the modified anti-TIGIT antibody (7A6 VH3/Vk5-DLE) was also tested for its cytotoxic ability against tumor cells. Tumor cells were counted using the Live-Cell analysis system (Satorius).

The obtained results are shown in Figure 16. As shown in Figure 16, the anti-TIGIT antibodies 7A6 VH3/Vk5 and 7A6VH3/Vk5-DLE of the present application both showed enhanced tumor cell killing effects.

6.3. Cytotoxicity of anti-TIGIT antibodies against ovarian cancer cells 1

To verify the cytotoxic capacity of anti-TIGIT 7A6 VH3/Vk5 antibodies against SKOV-3 tumor (ovarian cancer) cell line, SKOV-3 cells (35k cells/well) were co-cultured with PBMC and CytoStim (1/50) in the presence or absence of anti-TIGIT antibodies (10 μg/ml) or reference antibodies (10 μg/ml). The ratio of effector cells to target cells (E:T) was set to 6:1. The reference antibodies used were the anti-TIGIT antibodies Mereo/313M32, BMS/22G2, Genentech/4.1D3 and Arcus/TIG1 (see Reference Examples). The acquired data were analyzed by the Live-Cell Analysis System (Sartorius) and the IncuCyte native software.

The results are shown in Figure 17a (cell mortality rate during culture time) and Figure 17b (cell mortality rate after 84 days of culture). As shown in Figures 17a and 17b, it was observed that the anti-TIGIT 7A6VH3/Vk5 antibody increased the cytotoxicity of tumor cells, and this cytotoxicity was significantly higher than that of the reference antibody.

6.4. Cytotoxicity of anti-TIGIT antibodies against ovarian cancer cells 2

To test the cytotoxicity of anti-TIGIT antibodies (anti-TIGIT 7A6VH3/Vk5 and 7A6VH4/Vk4, 10 μg/ml each) against SKOV-3 ovarian cancer cells, SKOV-3 cells (35k cells/well) were co-cultured with isolated CD8+ T cells and Cytostim for 108 days in the presence or absence of anti-TIGIT antibodies. The ratio of effector cells to target cells (E:T) was set to 3:1. The group treated with pembrolizumab in the same manner served as a control. Data were analyzed using IncuCyte native software.

The results are shown in Figure 18. From the results, it can be seen that the anti-TIGIT 7A6VH3/Vk4 antibody exhibits enhanced cytotoxicity against tumor cells, and this cytotoxicity is significantly higher than that of pembrolizumab.

6.5. Enhancement of NK cell cytotoxicity against SKOV3 tumor cells by TIGIT-CD155 (PVR) blockade

NK cells were pre-stimulated with or without IL-15 (5 ng/ml) for 48 h. These NK cells were isolated from human PBMCs using an NK Cell Isolation Kit (from Miltenyi Biotec (Catalog No. 130-092-657)) according to the manufacturer's instructions. After culture, NK cells (250,000 cells/reaction) were co-cultured with SKOV3 cells (2.5×10e4 cells/reaction) in the presence of anti-TIGIT antibodies (7A6 VH3/Vk5, 7A6 VH4/Vk4, 7A6 VH3/Vk5-DLE) for 48 h, each antibody at a concentration of 10 μg/ml, and the E:T ratio was set to 10:1. Tumor cells were counted using the Live-Cell analysis system (Sartorius).

The results are shown in Figure 19. As shown in the figure, all tested anti-TIGIT antibodies significantly increased the cytotoxic capacity of NK cells against SKOV3 tumor cells.

Example 7: Analysis of in vivo anticancer efficacy

7.1. Determination of in vivo efficacy of anti-TIGIT antibodies in the MC38 colon cancer model

MC38 colon cancer cells (CrownBio) expressing human PVR (MC38-hPVR) were stored in vitro in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 4 μg/ml puromycin at 37°C in a 5% CO ₂ atmosphere. Before tumor inoculation, cells in the exponential growth phase were harvested and quantified using a cell counter.

The prepared tumor cell solution (2×10 ⁵ tumor cells in 0.1 mL PBS solution) was subcutaneously injected into the right side of C57BL6hTIGIT knock-in mice (from GemPharmatech Co., Ltd.) to induce tumor growth. Randomization was performed when the average tumor size (volume) reached 80 mm ³ to 100 mm ^3. A total of 15 mice were used in this experiment and randomly divided into three groups (5 mice/group).

After inoculation of tumor cells, the animals were checked for pathological status and mortality every day, and after randomization, the weight gain/loss was measured three times a week. The mortality rate and observed clinical symptoms of each animal were recorded daily. After randomization, the tumor size (volume) was measured in two dimensions using a caliper three times a week and calculated using the following formula:

V( ^mm3 )＝(L×W×W)/2

[Where V is the tumor volume (mm ³ ), L is the length of the tumor (the longest axis of the tumor), and W is the width (the longest length perpendicular to L)].

Inoculation, tumor size and weight measurements were performed in a laminar flow cabinet. Weight and tumor size were measured using StudyDirector ^™ software (version 3.1.399.19).

After grouping, the animals were immediately treated with antibodies or vehicle. PBS solution was used as vehicle (control; Group 1), and 7A6 VH3/Vk5 produced by CHO cells and tirilimumab were treated at a dose of 20 mg/kg (Groups 2 and 3, respectively) (see Table 13).

Table 13

BIW: Bi-weekly (twice a week)

i.p.: intraperitoneal injection

During the test period, no weight loss was observed in the groups injected with the antibody (Groups 2 and 3; dose 20 mg/kg, intraperitoneal injection).

The tumor sizes of mice measured as above are shown in FIG20 .

As shown in Figure 20, on day 21 after antibody treatment, the 7A6 VH3/Vk5 antibody showed a significant inhibitory effect on tumor growth compared to the PBS control group. When the size of the MC38 tumors between the matched day test groups was analyzed using an unpaired t-test, the tumor growth inhibitory effect of the 7A6 VH3/Vk5 antibody was particularly evident on day 9 (p value, 0.029) and day 21 (p value, 0.007) compared to the control group. The 7A6 VH3/Vk5 antibody (Group 2) also showed better anti-tumor efficacy than the control antibody tirilimumab (Group 1) (e.g., on day 21, p value was 0.013).

Tumor growth inhibition (ΔTGI) was calculated as the mean % Δ inhibition:

ΔTGI (average % Δ inhibition) = ((average (C) - average (C0)) - (average (T) - average (T0))) / (average (C) - average (C0)) * 100%

[T: tumor size of the test group at the measurement point,

T0: initial tumor size of the test group,

C: tumor size at the measurement point in the control group, and

C0: initial tumor size of the control group]

The tumor growth inhibition (TGI) of the 7A6 VH3/Vk5 antibody was 67.9%, showing enhanced inhibition compared to tisleliumab (TGI = 55.2%).

7.2. In vivo study on enhancing anti-tumor efficacy by combined treatment with anti-TIGIT and anti-PD1 antibodies in the CT26 colon cancer model

CT-26 tumor cells (CrownBio) were maintained in vitro in RPMI1640 medium supplemented with 10% FBS at 37°C in an atmosphere of 5% CO _2. Before inoculation of tumor cells, cells in the exponential growth phase were harvested and quantified using a cell counter.

The prepared tumor cell solution (5×10 ⁵ tumor cells in 0.1 mL PBS solution) was subcutaneously injected into the right hind flank of BALB/c hPD-1/hTIGIT double knock-in mice (GemPharmatech Co., Ltd.), and the tumor was allowed to grow. The day of tumor inoculation was designated as "Day 0". Randomization was performed when the average tumor size (volume) reached 70 mm ³ to 100 mm ^3. A total of 30 mice were used in this experiment and randomly divided into 5 groups (6 mice/group).

After inoculation of tumor cells, the body weights of the animals were measured.

PBS solution was used as a vehicle (control group; Group 1), treated with 7A6 VH3/Vk5 and 7A6 VH3/Vk5-DLE at a dose of 20 mg/kg (Groups 3 and 4, respectively), treated with the anti-PD1 antibody Keytruda (pembrolizumab; Merck) at a dose of 5 mg/kg (Group 4), and 7A6 VH3/Vk5 and Keytruda 5 mg/kg were administered in combination at doses of 20 mg/kg and 5 mg/kg (Group 5).

The experiment was terminated when the mean tumor burden of the vehicle-treated group (control group) reached 3000 mm ³ on day 25 (see Table 14).

Table 14

BIW: Bi-weekly (twice a week)

Calculate TGI% according to the following equation:

TGI (%) = 100 × (1-T/C)

(T and C represent the average tumor volume (or weight) of the experimental group (T) and the control group (C), respectively.

The results are summarized in Table 15 and depicted in Figure 21.

Table 15

As shown in Table 15 and Figure 21, on day 25, both the 7A6 VH3/Vk5-treated group (Group 3) and the Keytruda-treated group (Group 2) showed reduced tumor growth, with TGI (tumor growth inhibition) values of 51.43% and 55.91%, respectively. The combination of Keytruda and 7A6VH3/Vk5 (Group 5) showed the most significant delay and inhibition of tumor formation, with a TGI of 78.98%. On day 25, the tumor growth inhibition effect of all 7A6 VH3/Vk5-, Keytruda-, and Keytruda+7A6 VH3/Vk5-treated groups was statistically significantly higher than that of the control group (p value < 0.05). Complete tumor removal was observed in the 7A6 VH3/Vk5-treated group (1 mouse) and the Keytruda+7A6VH3/Vk5-treated group (2 mice). No significant body weight differences were observed in any antibody-treated group.

In addition, the effects on immune cells in the tumor microenvironment (TME) were detected, and the results are shown in FIG22 .

For FACS analysis, tumors were dissected from euthanized mice and dissociated into single cells using gentleMACS (GentleMACSTM Octo dissociator with heater) and multi-tissue dissociation kit 1 (Miltenyi Biotech). Cells were dyed with the following fluorescently labeled antibodies for surface markers, such as anti-mouse CD45 FITC (Biolegend), anti-mouse CD3-BUV395 (BD), anti-mouse CD4-BV421 (Biolegend), anti-mouse CD8-PE-eFluor610 (eBiosciences), anti-mouse CD335 (eBiosciences) and live/dead-APC-eF780 (eBiosciences). In addition, fixation/permeabilization concentration and dilution kit (ThermoFisher) were used to fix and permeabilize cells, and then anti-mouse Foxp3 (eBiosciences) was used for staining. Cells were analyzed by flow cytometer (LSRFortessa X-20, BD), and data were analyzed using FlowJo data analysis software.

As shown in FIG. 22 , combined administration of anti-TIGIT antibody and anti-PD1 antibody reduced the frequency of Tregs in tumors, resulting in an increase in CD3, CD4, and CD8+ T cells ( FIG. 22 ).

7.3. In vivo determination of the efficacy of anti-TIGIT antibodies in a humanized liver cancer mouse model with patient-derived tumor xenografts (PDX)

Human CD34+ hematopoietic stem cells (HSC) from umbilical cord blood of three donors (Jackson Laboratory, USA) were transplanted into immunodeficient NSG mice (NOD.Cg-Prkdscid Il2rgtm1Wj1/SzJ) (Jackson Laboratory, USA) for in vivo analysis. Hepatobiliary carcinoma LIXFC 2479 (Charles river) tumor cells were obtained from surgical specimens after resection of cancer patients. Tumor fragments were transplanted into immunodeficient mice at the first generation and cultured (passaged) as tumor xenografts until a stable growth pattern was established. Tumor fragments were obtained from nude mouse xenografts by serial passaging. After dissection from the donor mouse, the tumor was cut into slices (3 mm to 4 mm in circumference) and placed in PBS containing 10% penicillin/streptomycin. The slices were then transplanted subcutaneously into one side of the recipient mouse, which is called a patient-derived tumor xenograft (PDX) model.

These animals were divided into two groups, each consisting of six mice. Two mice from each group were reconstructed with HSC from each of the three donors to ensure similar median tumor volume (50 mm ³ to 150 mm ³ ) and weight at the beginning. The randomization day was set as "Day 0". PBS was used as a control (Group 1). The mice in Group 2 were treated with 7A6VH3/Vk5 at a dose of 20 mg/kg. The test substance was administered twice a week by intraperitoneal injection for four weeks, and tumor size was monitored until the end of the experiment on Day 28. Detailed information is shown in Table 16 below:

Table 16

*Based on last weight measurement

**Donor 1+Donor 2+Donor 3

BIW: Bi-weekly (twice a week), ROA: route of administration

Tumor growth inhibition (ΔTGI) was calculated as the mean % Δ inhibition:

ΔTGI (average % Δ inhibition) = ((average (C) - average (C0)) - (average (T) - average (T0))) / (average (C) - average (C0)) * 100%

[T: tumor size when measured in the test group,

T0: initial tumor size of the test group,

C: The size of the tumor when measured in the control group,

C0: initial tumor size of the control group]

The results obtained (tumor size) are shown in Figure 23. As shown in Figure 23, the anti-TIGIT 7A6VH3/Vk5 antibody significantly inhibited tumor growth compared with the control group. To evaluate TGI, the tumor size between the anti-TIGIT antibody-treated group and the control group was compared on day 28. The tumor growth inhibition (ΔTGI) of the 7A6 VH3/Vk5 antibody was 76.6%, indicating that the antibody has a significant anti-cancer effect in the liver cancer PDX tumor model, which is considered to have greater physiological relevance to human cancer than synthetic mouse models.

Example 8: Anti-tumor efficacy of anti-TIGIT antibodies in human PBMCs and tumor-infiltrating T cells

8.1. TIGIT expression on T cell subsets in the tumor microenvironment (TME)

To measure the potential role of anti-TIGIT antibodies in the human tumor microenvironment, the expression of TIGIT in the livers of HCC (hepatocellular carcinoma) patients and HFE (hemochromatosis) donors was detected. HFE (hemochromatosis) is considered to be a disease with normal liver function and immune health. In order to study the expression of TIGIT in T cell subsets, tumor infiltrating lymphocytes from normal donors and HCC cancer patients were prepared (see Example 3.1). These cells were stained with cell surface markers (CD3, CD4, CD8, CD127, CD25, TIGIT) (antibodies for CD3, CD4, CD8, CD127 and CD25 were obtained from Biolegend, and TIGIT antibodies were obtained from R&D Systems) and analyzed by flow cytometry and FlowJo software. The mean fluorescence intensity (MFI) of TIGIT in each T cell subset was measured using FlowJo software (BD Biosciences).

The results obtained are shown in Figure 24. As shown in Figure 24, TIGIT expression on tumor-infiltrating T cells in HCC patients was higher than that in normal donors. Among tumor-infiltrating T cells in HCC patients, Treg cells expressed the highest level of TIGIT compared with CD4+ and CD8+ T cells. These findings suggest that Treg cells may be an ideal target for the inhibitory activity of anti-TIGIT antibodies.

8.2. Cytokine production in T cells of patients with liver cancer

Immune cells expressing TIGIT can cause dysfunction in cancer patients. We tested the ability of anti-TIGIT antibody treatment to restore or enhance immune responses in HCC patients.

To measure anti-TIGIT antibody-mediated cytokine production from T cells, human PBMCs obtained from hepatocellular carcinoma (HCC) patients or healthy donors were cultured with anti-CD3 and anti-CD28 antibodies in the presence of anti-TIGIT 7A6VH3/Vk5 antibodies (10 μg/ml) or the same amount of reference antibodies (anti-TIGIT 10A7, 22G2). Intracellular cytokine production was measured (see Example 3.2.2).

The results obtained are shown in Figure 25. Treatment with anti-TIGIT 7A6 VH3/Vk5 antibodies increased cytokine production in CD4+ and CD8+ T cells from PBMCs of HCC patients compared to reference antibodies, anti-TIGIT antibodies 10A7 (Genentech) and 22G2 (BMS). Since IL-2 and IFN-γ are cytokines with core functions in the immune system, enhancing the proliferation and death activities of NK and T cells, the data obtained show the efficacy of anti-TIGIT 7A6 antibody treatment in HCC patients.

8.3. Efficacy of anti-TIGIT antibodies on T cells from lung cancer patients

Cytokine production in immune cells from human PBMCs from non-small cell lung cancer (NSCLC) patients was tested after treatment with anti-TIGIT antibodies (10 μg/ml each) (see Example 3.2.2).

The results obtained are shown in Figure 26. Compared with the reference antibodies, anti-TIGIT antibodies 10A7 (Genentech) and 22G2 (BMS), anti-TIGIT 7A6 VH3/Vk5 antibodies increased the cytokine production of CD4+ and CD8+ T cells in PBMCs of patients with non-small cell lung cancer to a higher level. These results confirm the efficacy of anti-TIGIT 7A6 antibodies in patients with non-small cell lung cancer.

8.4. Efficacy of anti-TIGIT antibodies on T cells from patients with colorectal cancer

Immune cells expressing TIGIT induce functional impairment in cancer patients, and an examination was conducted to investigate whether treatment with anti-TIGIT antibodies could restore or enhance the immune response in patients with colorectal cancer. Cytokine production in immune cells from human PBMCs of patients with colorectal cancer (CRC) was tested after treatment with anti-TIGIT antibodies (10 μg/ml each) (see Example 3.2.2).

The results obtained are shown in Figure 27. Compared with the reference antibodies, anti-TIGIT antibodies 10A7 (Genentech) and 22G2 (BMS), the anti-TIGIT 7A6 VH3/Vk5 antibody increased the cytokine production of CD4+ and CD8+ T cells in PBMCs of colorectal cancer patients to a higher level. These results indicate the efficacy of anti-TIGIT 7A6 antibodies in colorectal cancer patients.

Example 9: Assaying T cell activation by inhibition of TIGIT-PVR interaction with anti-TIGIT Fab fragments

9.1. Immune effects of anti-TIGIT Fab on central memory T cells and effector memory T cells

To verify the specific blocking effect of anti-TIGIT 7A6 VH3/Vk5 antibody on TIGIT/PVR pathway, the anti-TIGIT-Fab fragment (disulfide bond between 7A6_VH3 variable region and 7A6_VK5 variable region) of the antibody was produced in HEK293 cells and purified using immobilized metal affinity chromatography. The anti-TIGIT Fab fragment was used to exhibit only specific blocking function without Fc-induced effect.

Through the specific binding of anti-TIGIT Fab fragments to TIGIT protein, central memory T cells [Tcm] (Figure 28a) and effector memory T cells [Tem] (Figure 28b) were detected, which provides long-term and effective anti-cancer cell toxicity protection. Specifically, 500,000 PBMC cells were activated with anti-CD3 (0.2 μg/ml) (BD Biosciences) and anti-CD28 antibodies (1 μg/ml) (BD Biosciences), and then treated with anti-TIGIT Fab fragments (10 μg/ml) for 48 hours. Brefeldin A (Sigma) and Monensin (Sigma) were added to each well to a final concentration of 1/1000 and incubated for another 4 hours. Central memory T cells and effector memory T cells were stained. In this regard, cells were washed and stained at room temperature for 10 minutes with the following antibodies: Alive/Dead (ThermoFisher LIVE/DEAD ^TM Fixable Near-IR Dead CellStain Kit, for 633nm or 635nm excitation), anti-CD3-APC (Biolegend), anti-CD8-PerCP (Biolegend), anti-CD45-RA-Brillant Violet 421 (Biolegend), anti-CCR 7-Brilliant Violet 510 (Biolegend). After staining, the cytokine levels in central memory T cells and effector memory T cells were measured as follows. According to the manufacturer's instructions, Foxp 3/transcription factor fixation/permeabilization concentrate and diluent kit (ThermoFisher) were used to fix and permeabilize cells. Cells were stained with fluorophore-conjugated antibodies for intracellular IL-2, IFNγ and TNFa (all from Biolegend). Cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter), and data were analyzed using FlowJo software (BD Biosciences).

The results obtained are shown in Figure 28a (central memory T cells) and Figure 28b (effect memory T cells). From the results, it can be seen that the anti-TIGIT-Fab fragment induced enhanced activation levels of Tcm and Tem, proving that the anti-TIGIT (7A6 VH3/Vk5) antibody mediates specific blocking activity and promotes long-term immune response against cancer.

9.2. Anti-TIGIT Fab fragment-mediated cytotoxic stimulation of A375 tumor cells

A375 tumor cells (35k cells/well) were stained with Syto-9 dye and co-cultured with PBMC and CytoStim (1/50) in the presence of Fab fragments generated by humanized anti-TIGIT antibodies (Fab 7A6 VH0/Vk0, 3VH3/Vk5, VH4/Vk4) (10 μg/ml each). Tumor cells were counted using the Live-Cell analysis system (Sartorius).

The results are shown in Figure 29. All tested anti-TIGIT Fab fragments VH0/Vk0, VH3/Vk5 and VH4/Vk4 blocked TIGIT-CD155 (PVR) interaction and increased cytotoxicity against A375 tumor cells. These results confirm that humanized anti-TIGIT 7A6 antibodies effectively blocked the TIGIT-CD155 (PVR) pathway and induced enhanced immune function.

Example 10: Immunomodulatory effects of anti-TIGIT antibodies on regulatory T cells and NK cells

10.1. High Level Expression of TIGIT on Regulatory T Cells (Tregs)

PBMCs from healthy donors were stained for markers of T cell subsets and TIGIT protein (anti-CD3-APC (Biolegend), Alive/Dead (ThermoFisher LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit), anti-CD8-PerCP (Biolegend), anti-CD4-Alexa Fluor700 (Biolegend), anti-CD127-PE (Biolegend), anti-CD25-Brilliant Violet 421 (Biolegend), and anti-TIGIT-PeCy7 (Biolegend)). After staining, cells were centrifuged and analyzed by flow cytometry.

The results are shown in Figure 30. TIGIT was expressed at a higher level in regulatory T cells (Treg) compared to non-Treg CD4+ and CD8+ T cells.

10.2. Treg depletion activity of anti-TIGIT 7A6 VH3/Vk5 antibodies

Anti-TIGIT 7A6 VH3/Vk5 antibody (10 μg/ml) was co-cultured with human PBMCs in the presence of anti-CD3 antibody (BD Biosciences) and anti-CD28 antibody (BD Biosciences). Anti-TIGIT 10A7 (Genentech) was used as a control. The frequency of Treg, CD4+T and CD8+T cells was assessed by flow cytometry.

The results obtained are depicted in Figures 31a (Treg), 31b (CD4+T cells), 31c (CD8+T cells) and 31d. The anti-TIGIT 7A6VH3/Vk5 antibody specifically targets Treg cells without changing the ratio of CD4+ and CD8+T cells. In addition, compared with the control anti-TIGIT antibody (10A7), the anti-TIGIT antibody depleted Treg at a higher level (Figure 31d).

10.3. NK cell-mediated Treg depletion activity of anti-TIGIT 7A6 VH3/Vk5 antibodies

Treg and NK cells were co-cultured overnight in the presence or absence of anti-TIGIT antibodies 7A6 VH3/Vk5 or VH3/Vk5-DLE (10 μg/ml each). The next day, the remaining Treg and NK cells were counted by flow cytometry. Treg cells and NK cells were isolated from human PBMCs using the Miltenyi kit (CD4+CD25+CD127-dim reg T cell isolation kit II, catalog number 130-094-775) and the Miltenyi kit (NK cell isolation kit, catalog number 130-092-657), respectively, according to the manufacturer's instructions.

The results obtained are shown in Figure 32. After treatment with anti-TIGIT 7A6VH3/Vk5 antibody and VH3/Vk5-DLE antibody, the number of Treg cells decreased, while the number of NK cells remained unchanged. These results indicate that the tested anti-TIGIT antibodies effectively deplete Tregs through antibody-dependent cellular cytotoxicity and NK cells.

In addition, we examined whether Fc receptor binding is required for NK cell-mediated Treg elimination by anti-TIGIT antibodies.

Treg and NK cells were co-cultured overnight in the presence of anti-TIGIT antibody 7A6 VH3/Vk5-DLE (10 μg/ml). According to the manufacturer's instructions, Treg cells and NK cells were isolated from human PBMC using Miltenyi kit (CD4+CD25+CD127-dim reg T cell isolation kit II, catalog number 130-094-775) and Miltenyi kit (NK cell isolation kit, catalog number 130-092-657). Fc receptors were blocked using Fc blocking antibody CD16 and Human TruStain FcX ^TM (BioLegend). The next day, the remaining Treg and NK cells were counted by flow cytometry.

The results obtained are shown in Figure 33. Anti-TIGIT antibodies significantly reduced Tregs, and this reduction was inhibited when FcR was blocked by blocking antibodies. These results indicate that Fc receptor binding is required for NK cell-mediated Treg depletion by anti-TIGIT antibodies.

10.4. Fc-dependent T cell activation

Cytokine production in human T cells (CD4+T cells and CD8+T cells) was measured with or without pre-blocking FcgRIIIA. 500,000 PBMC cells were pre-incubated with FcgRIIIA antibody (Biolegend catalog number 422302) at room temperature for 5 minutes. PBMC cells were then cultured with 0.2μg/ml anti-CD3 (BD Biosciences) and 1μg/ml anti-CD28 (BD Biosciences) monoclonal antibodies in the presence of anti-TIGIT 7A6VH3/Vk5 (10μg/ml) or isotype control antibody (10μg/ml). After culture, cells were centrifuged at 400xg for 10 minutes at 4°C, and intracellular cytokine concentrations in T cells were measured. In this regard, cells were stained with ZombieAqua fixable dead cell dye solution (Biolegend), and labeled on ice for 30 minutes with fluorophore-conjugated antibodies (Biolegend) for CD3, CD4 and CD8+ cell surface markers for CD4+ and CD8+T cells. According to the manufacturer's instructions, eBioscience ^TM Foxp3/ transcription factor fixation/permeabilization concentration and dilution kit (ThermoFisher) was used to fix and permeabilize cells. Cells were stained with fluorophore-conjugated antibodies for intracellular IL-2 (Biolegend) and IFNγ (Biolegend). Cells were then analyzed using a flow cytometer (CytoFLEX, Beckman Coulter), and data were analyzed using FlowJo software (BD Biosciences).

The results obtained are depicted in Figures 34a (CD4+T cells) and 34b (CD8+T cells). Blockade of FcgRIIIA significantly reduced cytokine production in CD4+ and CD8+T cells, indicating that anti-TIGIT antibodies require FcgR interaction to activate T cells and deplete Tregs. Anti-TIGIT antibodies enhance T cell responses through the engagement of FcgR.

statistics

Statistical significance was assessed by ordinary one-way ANOVA, paired and unpaired t-test using GraphPad Prism 9 software (GraphPad Software, USA). Data are presented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant, as indicated in the figure legend.

Claims (14)

1. An anti-TIGIT antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody comprises: A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 2, a polypeptide (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 3, A polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (CDR-L1), A polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and A polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3). 2. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-TIGIT antibody comprises: A polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CDR-H1), a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 2, A polypeptide (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 3, a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, A polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and A polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (CDR-L3). 3. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-TIGIT antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20. 4. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof binds to at least one amino acid selected from amino acids 51 to 70 in the amino acid sequence of the TIGIT protein. 5 . The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-TIGIT antibody is an animal antibody, a chimeric antibody or a humanized antibody. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is scFv, scFv-Fc, (scFv)2, Fab, Fab' or F(ab') ₂ of the anti-TIGIT antibody. 7. A pharmaceutical composition for preventing or treating cancer, comprising the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. 8. The pharmaceutical composition according to claim 7, wherein the cancer is a solid cancer or a blood cancer. 9. The pharmaceutical composition according to claim 7, further comprising a drug targeting PD-1, PD-L1 or both. 10 . A pharmaceutical composition for immune enhancement, comprising the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. 11. The pharmaceutical composition according to claim 10, wherein the composition further comprises a drug targeting PD-1, PD-L1 or both. 12. A pharmaceutical composition for preventing or treating immune-related diseases, comprising the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. 13. The pharmaceutical composition according to claim 12, wherein the immune-related disease is an infectious disease, an inflammatory disease or an autoimmune disease. 14. The pharmaceutical composition according to claim 12, further comprising a drug targeting PD-1, PD-L1 or both.